Schizosaccharomyces

Fungal Genet Biol, 1998 Jun-Jul, 24(1-2), 178 - 206
Dynamics of cell wall formation in fission yeast, Schizosaccharomyces pombe; Osumi M et al.; Studies on the dynamics of surface and intracellular structures during cell wall formation from the reverting protoplast of Schizosaccharomyces pombe were reviewed, and the correlation between cell wall formation and actin cytoskeleton, which is the most important conductor of the mechanism, is described in this paper . A close spatial and temporal relationship between actin cytoskeleton and cell wall formation was found by using wild type and actin point-mutant cps8 of S . pombe . Concomitant with the cell wall formation, dynamic behavior of the intracellular secretion machinery, especially the Golgi apparatus and secretory vesicles, was analyzed by three-dimensional reconstruction of 40 to 80 serial sections at five reverting stages . Total reverting protoplast volume increased by 3.8 and 4.3 times at 3 and 5 h, respectively, and the volume of the Golgi apparatus in the corresponding stages increased 2.3- and 2 . 5-fold over the same periods . The number of secretory vesicles also markedly increased by 3.4 and 5.8 times over that of the corresponding reverting protoplasts . Actin point-mutant cps8 cells have abnormal structure in the cell wall and septum, and the distribution pattern of the actin cytoskeleton during the reversion process was different from wild-type protoplasts . The profiles of actin showed one or two thick cables and patches in the cytoplasm which remained throughout reversion . The development of crosslinkage of the glucan fibrils which are beta-1,3-glucan in nature on the reverting protoplast surface was defective; the glucan networks consisted of thin, rope-shaped fibrils up to 30 nm in width which formed a ribbon-shape 200 nm wide in wild-type reverting protoplasts . The intrafibrillar space is not filled with amorphous particles of alpha-galactomannan in nature . The secretion machinery was seen to have a similar profile as the wild type . The above results suggest that actin cytoskeleton may control secretion of beta-1,6-glucan and other cell wall substances such as alpha-glucan and alpha-galactomannan rather than beta-1,3-glucan . Study of the role of actin cytoskeleton in the cell wall formation is contributing to the development of antifungal agents together with basic cell biology .

Curr Biol, 1998 Sep 10, 8(18), 1023 - 6
A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast; Osmulski PA et al.; One eukaryotic proteolytic complex--the proteasome--is classed as the major nonlysosomal protease, by its known and suspected functions, its size and its complexity . It seems improbable that other enzymes may be capable of substituting, even partially, for the potent proteasome, as this complex has a vital role in many cellular processes . Nevertheless, it is possible to adapt cultured EL-4 mouse lymphoma cells to survive in the presence of a specific inhibitor of the proteasome . The inhibition of the proteasome in these adapted EL-4 cells is accompanied by a dramatic increase in the activity of a new, as yet uncharacterized, large proteolytic complex . Here, we have presented evidence that a similar proteolytic activity is constitutively present in fission yeast, Schizosaccharomyces pombe, and that the yeast and mouse enzymes share basic physicochemical properties . We have shown that the S . pombe protease is found in two stable oligomeric forms, both of which are peptidases, although only the larger form acts as a proteinase . The relative amounts of the large and the small forms of the protease in the complex depended on the growth phase of the yeast culture and affected enzyme activity, suggesting that the activity of the enzyme is regulated by its oligomerization status . We refer to the new proteolytic complex as the 'multicorn' to indicate its analogy to the archaebacterial tricorn protease.

J Cell Sci, 1998 Oct, 111 ( Pt 20), 3101 - 8
Cdc18p can block mitosis by two independent mechanisms; Greenwood E et al.; The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed . The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis . The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle . We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p . The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis . The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes . Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

Mol Gen Genet, 1998 Jul, 259(1), 123 - 9
Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast; Miyao T et al.; {Rpb1 and Rpb2} Mapping of the contact sites on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae . Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the beta' subunit of prokaryotic RNA polymerases . In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5 . On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the beta subunit of prokaryotic RNA polymerases . Possible functions of the regions H in the subunits 1 and 2 are discussed.

J Biol Chem, 1998 Sep 25, 273(39), 25098 - 105
Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from yeast Schizosaccharomyces pombe; Lu AL et al.; The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombe encodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively . Expressed SpMYH is able to complement an E . coli mutY mutant to reduce the mutation rate . Similar to E . coli MutY protein, purified recombinant SpMYH expressed in E . coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA . However, both enzymes have different salt requirements and slightly different substrate specificities . SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C than E . coli MutY . Both enzymes also have different substrate binding affinity and catalytic parameters . Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY . SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA . Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect.

Hum Mol Genet, 1998 Oct, 7(11), 1703 - 12
Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human; Laporte J et al.; X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene . The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases . Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine . This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family . We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man . This represents the largest family of DSPs described to date . Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them . A subclass of the myotubularin homologues lacks a functional PTP active site . Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue . Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function . These genes may be good candidates for other genetic diseases.

Genes Cells, 1998 Jun, 3(6), 347 - 55
Defect in cytokinesis of fission yeast induced by mutation in the WD40 repeat motif of a TFIID subunit; Yamamoto T et al.; BACKGROUND: TBP-associated factors contain a variety of structural motifs and their related in vivo significance has remained unclear . We have attempted to identify specific biological phenomena linked to a particular domain of a TAF by analysing domain-exchanged chimeric mutants between Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc) counterparts . RESULTS: Contrary to the case of TBP, Sp TAF containing the WD40 repeat cannot be exchanged for its Sc counterpart, despite their highly conserved primary structures . This 'species-specific' function locates in the N-terminal region . The C-terminal region, largely consisting of the WD40 repeat, is exchangeable for the corresponding region of its Sc counterpart . Growth of the strain harbouring this C-terminal chimeric mutant is temperature-sensitive . The chimeric gene product did not disappear at a restrictive temperature, a finding which strongly suggests that the growth defect is caused by an aberration in the interactions through the WD40 repeat structural motif . With temperature elevation, the chimeric mutants underwent drastic morphological changes due to a defect in cytokinesis . CONCLUSIONS: The WD40 repeat of TAF is primarily involved in reactions which might regulate cytokinesis in Sp.

Yeast, 1998 Aug, 14(11), 1051 - 9
The uracil permease of Schizosaccharomyces pombe: a representative of a family of 10 transmembrane helix transporter proteins of yeasts; de Montigny J et al.; The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced . The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w . A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S . cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters . The sequence of the uracil permease gene of S . pombe has been deposited in the EMBL data bank under Accession Number X98696.

Biochem J, 1998 Sep 15, 334 ( Pt 3), 609 - 16
Human and mouse Gpi1p homologues restore glycosylphosphatidylinositol membrane anchor biosynthesis in yeast mutants; Tiede A et al.; Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins . The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI) . The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex . Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins . To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains . In Sacch . cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner . In addition, in vitro GlcNAc-PI synthetic activity was again detectable . In Schiz . pombe, gpi1+ disruption is lethal . Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele . Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex . The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution.

Biochim Biophys Acta, 1998 Aug 24, 1381(3), 271 - 8
Trehalose-6P synthase is essential for trehalase activation triggered by glucose, nitrogen source or heat shock, but not by osmostress, in Schizosaccharomyces pombe; Cansado J et al.; Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source . Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock . Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock . In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar . Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells . Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells . These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock . However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz . pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress . Copyright Elsevier Science B.V.

J Biol Chem, 1998 Sep 11, 273(37), 23938 - 45
The Pad1+ gene encodes a subunit of the 26 S proteasome in fission yeast; Penney M et al.; We have isolated a fission yeast mutant, mts5-1, in a screen for mutations that confer both methyl 2-benzimidazolecarbamate resistance (MBCR) and temperature sensitivity (ts) on Schizosaccharomyces pombe . This screen has previously isolated mutations in the 26 S proteasome subunits Mts2, Mts3, and Mts4 . We show that the mutation in the mts5-1 strain occurs in the pad1(+) gene . pad1(+) was originally isolated on a multicopy plasmid that was capable of conferring staurosporine resistance on a wild type strain . mts5-1/pad1-1 has a similar phenotype to 26 S proteasome mutants previously isolated in the same screen and we show that Pad1 interacts genetically with two of these subunits, Mts3 and Mts4 . In this study we describe the identification of Pad1 as a subunit of the 26 S proteasome in fission yeast.

Nucleic Acids Res, 1998 Sep 15, 26(18), 4222 - 9
The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin; Doe CL et al.; In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided . The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin . Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function . We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ) . In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4 . A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus . In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes . We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)) . Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.

Microbiology, 1998 Aug, 144 ( Pt 8), 2331 - 44
Cytochalasin D interferes with contractile actin ring and septum formation in Schizosaccharomyces japonicus var . versatilis; Gabriel M et al.; The cells of Schizosaccharomyces japonicus var . versatilis responded to the presence of cytochalasin D (CD), an inhibitor of actin polymerization, by the disappearance of contractile actin rings (ARs) that had already formed and by inhibition of new ring formation . Actin cables disappeared . Actin patches remained preserved and became co-localized with regions of actual cell wall formation (at cell poles and at the site of septum development) . Removal of the AR arrested formation of the primary septum and led to the production of aberrant septum protrusions in that region . Nuclear division was accomplished in the presence of CD but new ARs were not produced . The wall (septum) material was deposited in the form of a wide band at the inner surface of the lateral cell wall in the cell centre . This layer showed a thin fibrillar structure . The removal of CD resulted in rapid formation of new ARs in the equatorial region of the cells . This implies that the signal for AR localization was not abolished either by CD effects or by removal of an AR already formed . Some of the newly developed ARs showed atypical localization and orientation . In addition, redundant, subcortically situated actin bundles were produced . The removal of CD was quickly followed by the development of primary septa co-localized with ARs . Wall protrusions occurred co-localized with the redundant actin bundles . If these were completed in a circle, redundant septa developed . The AR is a mechanism which, in time and space, triggers cytokinesis by building a septum sequentially dependent on the AR . Aberrant septa were not capable of separating daughter cells . However, non-separated daughter cells subsequently gave rise to normal cells.

Yeast, 1998 Jul, 14(10), 953 - 61
Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae; Longtine MS et al.; An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes . This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest . We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications . Using as selectable marker the S . cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging {with GFP(S65T), GST, or the 3HA or 13Myc epitope}, and partial N- or C-terminal deletions (with or without concomitant protein tagging) . Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations . Thus, these plasmids should further facilitate the rapid analysis of gene function in S . cerevisiae.

Yeast, 1998 Jul, 14(10), 943 - 51
Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe; Bahler J et al.; We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe . Using this approach and the S . pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63% . We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S . pombe . The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging . Nine genes were manipulated using these kanMX6 constructs as templates for PCR . The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome . Transformants were screened for homologous integration by PCR . In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17% . The methodology and constructs described here should greatly facilitate analysis of gene function in S . pombe.

Antonie Van Leeuwenhoek, 1998 Feb, 73(2), 189 - 94
Deleterious effects of androstenedione on growth and cell morphology of Schizosaccharomyces pombe; Dlugonski J et al.; Androgens (androstenedione and testosterone) belong to the most important compounds in human steroidogenesis . The 17 beta-hydroxysteroid dehydrogenase responsible for interconversion of the oxygenic group on C-17 of androgens ring is involved in steroid hormone synthesis . The fission yeast Schizosaccharomyces pombe 972 h- was found to contain constitutive 17 beta-hydroxysteroid dehydrogenase that was able to reduce androstenedione to testosterone and oxidize testosterone to androstenedione . The reductive pathway was found to be predominant while the oxidative one was carried out with much lower activity . Exogenous androstenedione, contrary to testosterone, inhibited S . pombe growth and stimulated the formation of aberrant swollen cells with slighter cell wall sensitivity to the action of the lytic enzyme Novozym . It is postulated that the 17 beta-hydroxysteroid dehydrogenase prevents the deleterious effects of androstenedione on the morphology and growth of the yeast's cells by androstedione reduction to testosterone.

Biochem J, 1998 Sep 1, 334 ( Pt 2), 377 - 86
Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5'-to-3' direction; Lee C et al.; We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe . The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity . Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel . The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 A (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa . The enzyme translocates in a 5'-to-3' direction with respect to the substrate strand to which it is bound . Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP . This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP . Non-hydrolysable ATP analogues did not support helicase activity . Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50-100 s of incubation . Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding . The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor . In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5-10 min in the presence of shorter oligonucleotides . The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used . These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive.

Genes Dev, 1998 Aug 15, 12(16), 2560 - 73
Human and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis; Freire R et al.; Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells . Several genes involved in this process have been discovered in fission and budding yeast . Here, we report the identification of human and mouse homologs of the Schizosaccharomyces pombe DNA damage checkpoint control gene rad1(+) and its Saccharomyces cerevisiae homolog RAD17 . The human gene HRAD1 is located on chromosome 5p13 and is most homologous to S . pombe rad1(+) . This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues . This human gene significantly complements the sensitivity to UV light of a S . pombe strain mutated in rad1(+) . Moreover, HRAD1 complements the checkpoint control defect of this strain after UV exposure . In addition to functioning in DNA repair checkpoints, S . cerevisiae RAD17 plays a role during meiosis to prevent progress through prophase I when recombination is interrupted . Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1 . Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans.

Gene, 1998 Jul 30, 215(2), 319 - 28
Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator; Hoe KL et al.; As a first step to elucidate the functions of Schizosaccharomyces pombe (S . pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S . pombe . The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1 . The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids . Disruption of this gene has no effect on cell viability and growth rate . The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S . cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain . These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.

Biochim Biophys Acta, 1998 Jul 30, 1399(1), 67 - 72
A Schizosaccharomyces pombe gene encoding a novel polypeptide with a predicted alpha-helical rod structure found in the myosin and intermediate-filament families of proteins; Jannatipour M et al.; We have identified a Schizosaccharomyces pombe gene encoding a 461 amino acid polypeptide containing a predicted alpha-helical rod domain found in filamentous proteins . This gene, here designated noc1, is located immediately upstream from cnx1, the gene encoding the S . pombe homologue of mammalian calnexin, an endoplasmic reticulum chaperone {M . Jannatipour, L.A . Rokeach, J . Biol . Chem . 270 (1995) 4845-4853.} . Transcription of noc1 is divergent from that of cnx1 . Northern blot analysis identified a single mRNA of approx . 2 kb whose expression was increased by heat shock and growth in the presence of beta-mercaptoethanol and 2-deoxyglucose.

Mol Cell Biol, 1998 Sep, 18(9), 5511 - 22
A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast; Singh J et al.; Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe . Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells . We have been interested in isolating mutants that are defective in this hypothesized coupling . An S . pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S . pombe as well as of S . cerevisiae . This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed) . Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase . Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6 . RAD6, an rhp6 homolog in S . cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B . This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination . We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.

Mol Cell Biol, 1998 Sep, 18(9), 5239 - 46
The role of fnx1, a fission yeast multidrug resistance protein, in the transition of cells to a quiescent G0 state; Dimitrov K et al.; Most microorganisms live in conditions of nutrient limitation in their natural habitats . When exposed to these conditions they respond with physiological and morphological changes that enable them to survive . To obtain insights into the molecular mechanisms of this response a systematic genetic screen was performed to identify genes that when overexpressed can induce a starvation-like response in the yeast species Schizosaccharomyces pombe . One gene that meets these criteria, fnx1(+), induces, transcriptionally correlates with, and is required for the entry into the quiescent G0 state that is normally induced by nitrogen starvation . fnx1(+) encodes a protein with sequence similarity to the proton-driven plasma membrane transporters from the multidrug resistance group of the major facilitator superfamily of proteins . We propose that fnx1(+) plays a role in the entry into G0, possibly by facilitating the release of a signaling substance into the environment as a means of cell-to-cell communication.

Biochemistry, 1998 Aug 18, 37(33), 11599 - 604
Expression, purification, and characterization of ultraviolet DNA endonuclease from Schizosaccharomyces pombe; Kaur B et al.; Ultraviolet damage endonuclease (UVDE) is a 68.7 kDa DNA repair enzyme of Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) . UVDE is thought to initiate the first step in an alternative excision repair pathway for removal of UV light-induced DNA damage . We have overexpressed Delta228-UVDE, an active truncated form of UVDE, and have purified this protein to apparent homogeneity . We have characterized purified Delta228-UVDE with respect to its physical properties, divalent cation requirements, and kinetic parameters on oligodeoxynucleotide substrates containing a single CPD . DNA strand cleavage analysis indicates that both full-length UVDE and Delta228-UVDE incise the CPD-containing strand immediately 5' to the lesion . These results provide further insight into the UVDE-mediated alternative excision repair pathway.

Nucleic Acids Res, 1998 Sep 1, 26(17), 3955 - 60
Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p); Sherman DA et al.; MCM proteins are required for the proper regulation of DNA replication . There are six MCM proteins in all eukaryotes which interact to form a large complex . We report the cloning of fission yeast mcm3 + . mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content . The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle . Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins . Thus, Mcm3p is a peripheral MCM subunit.

J Mol Biol, 1998 Jul 31, 280(5), 811 - 21
Site-specific insertion of a SINE-like element, Cp1, into centromeric tandem repeats from Chironomus pallidivittatus; Liao C et al.; A SINE-like dispersed element, Cp1, from the dipteran Chironomus pallidivittatus was found to show site-specific insertion into two different centromeric tandem repeats . The insertions result in identical target site duplications of nine base-pairs . In contrast, extracentromeric Cp1 elements, which are polymorphic and degenerate, are previously known to be surrounded by different target site duplications . The intracentromeric Cp1 is uniform in structure and contains a single pol III unit, upstream of which 87 bp arms of a palindrome surround a 103 bp unique sequence . The numbers of Cp1 elements per centromere were determined in microdissected material and were found to be in the range of five to ten units per centromere . The well-defined insertion properties, correlated to chromosomal localization, suggest that Cp1 is likely to be a component of importance for the centromere . Similarities of Cp1 and its parts to functionally identified centromeres in Saccharomyces cerevisiae and Schizosaccharomyces pombe are discussed .

RNA, 1998 Aug, 4(8), 958 - 72
The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast; Ito K et al.; Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3 . The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis . In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1 . The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35) . All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids . To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae . These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.

Trends Cell Biol, 1998 Apr, 8(4), 163 - 7
Discovering the poles in yeast; Mata J et al.; How cells generate and orientate polarized growth is of fundamental importance to understanding cell morphogenesis . The budding yeast Saccharomyces cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe have both been used for genetic analysis of cell morphogenesis . Generation and maintenance of their cell shape require the formation of polarized growth sites and the correct localization of these growth sites on the cell surface with respect to other cellular structures . In this review, the authors discuss and compare the mechanisms used by the two yeasts to achieve polarized growth.

Trends Cell Biol, 1998 Apr, 8(4), 144 - 9
Fission yeast cut mutations revisited: control of anaphase; Yanagida M; Studies of anaphase are approaching a golden age . Several different disciplines have contributed immensely to advances in our understanding of cell-cycle control and chromosome and spindle dynamics during mitosis . This article describes control of anaphase based on results obtained from Schizosaccharomyces pombe cut (cell untimely torn) mutants . These temperature-sensitive mutants were isolated by selection for uncoordinated mitosis with aberrant sister-chromatid separation and post-anaphase events . Characterization of some of the cut gene products has led to identification of novel molecular events related to chromosome condensation, sister-chromatid separation, anaphase-promoting proteolysis, fatty-acid metabolism, and cell-cycle arrest induced by stress or a replication block.

Mol Biol Cell, 1998 Aug, 9(8), 2325 - 35
The fission yeast mitotic regulator win1+ encodes an MAP kinase kinase kinase that phosphorylates and activates Wis1 MAP kinase kinase in response to high osmolarity; Samejima I et al.; The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle . Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known . We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1 . Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions . Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations . The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain . We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.

Mol Biol Cell, 1998 Aug, 9(8), 2231 - 47
The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane; Romano JD et al.; Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation . We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast . In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay . We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast . In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins . Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face . Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi . We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S . cerevisiae and have shown that mam4p complements a Deltaste14 mutant . This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

Mol Biol Cell, 1998 Aug, 9(8), 2011 - 23
The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains; Sicard H et al.; Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe . We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure . Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain . These drastic functional defects correlate with striking nucleolar hypertrophy . Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein . Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein . We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them . These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Protein Expr Purif, 1998 Aug, 13(3), 403 - 13
Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex; Niwa H et al.; We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms . The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae) . This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al . (S . Saitoh et al., 1996, J . Cell Biol . 134, 949-961), carries three domains . We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells . The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification . Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules . These results indicate that the FAS complex from S . pombe forms a heterododecameric alpha6beta6 structure . Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture .

Genetics, 1998 Aug, 149(4), 1753 - 61
Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus; Murata Y et al.; A homokaryotic strain (5337) in our culture stock of Coprinus cinereus produced fertile fruit bodies after prolonged culture . Microscopic examination revealed that hyphae dedifferentiated from the tissues of one of the fruit bodies, as well as all basidiospore derivatives from the fruit body, exhibited pseudoclamps, whereas vegetative hyphae of 5337, from which the fruit body developed, had no clamp connections . Genetic analysis showed that the formation of pseudoclamps results from a recessive mutation in a gene designated pcc1 (pseudoclamp connection formation), which is distinct from the A and B mating type genes . Cloning and sequencing of the pcc1 gene and cDNA identified an ORF of 1683 bp interrupted by one intron . Database searches revealed that pcc1 encodes an SRY-type HMG protein . The HMG box shared 44, 41, and 29% sequence identities (>80 amino acids) to those of FPR1 of Podospora anserina, MAT-Mc of Schizosaccharomyces pombe, and prf1 of Ustilago maydis, respectively . Northern analysis revealed that the level of pcc1 expression is higher in the dikaryon, in homokaryons in which the A and B mating type developmental sequences are individually activated, than in the homokaryon in which these sequences are not active . Sequencing of the pcc1-1 mutant allele revealed that the mutant carries a nonsense mutation at serine 211, a residue located between the HMG box and the C terminus . Based on these results, possible roles of the pcc1 gene in the sexual development of homobasidiomycetes are discussed.

Genetics, 1998 Aug, 149(4), 1729 - 37
The Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage; Rhind N et al.; We have identified an S-phase DNA damage checkpoint in Schizosaccharomyces pombe . This checkpoint is dependent on Rad3, the S . pombe homolog of the mammalian ATM/ATR checkpoint proteins, and Cds1 . Cds1 had previously been believed to be involved only in the replication checkpoint . The requirement of Cds1 in the DNA damage checkpoint suggests that Cds1 may be a general target of S-phase checkpoints . Unlike other checkpoints, the S . pombe S-phase DNA damage checkpoint discriminates between different types of damage . UV-irradiation, which causes base modification that can be repaired during G1 and S-phase, invokes the checkpoint, while gamma-irradiation, which causes double-stranded breaks that cannot be repaired by a haploid cell if induced before replication, does not invoke the checkpoint . Because the same genes are required to respond to UV- and gamma-irradiation during G2, this discrimination may represent an active suppression of the gamma response during S-phase.

EMBO J, 1998 Aug 3, 17(15), 4503 - 10
A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast; Arcangioli B; Mating-type switching in the fission yeast Schizosaccharomyces pombe results in the transfer of genetic information from one of the two silent cassettes (mat2P or mat3M) to the transcriptionally active locus (mat1) . The switching pattern is programmed by an imprinting event which restricts mat1 gene conversion to only one of the two sister cells, leading to asymmetric cell division . Biochemical analysis indicated that the mat1 locus contains a fragile chromosomal site . Southern hybridization and primer extension experiments showed that the fragility consists of a single-strand break (SSB) . The nicked DNA is stable throughout the cell cycle . The features of the nick fulfil all the requirements for the 'epigenetic', site and strand-specific chromosome modification at the mat1 locus, providing strong evidence that an SSB can initiate mitotic and meiotic gene conversion during replication.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3762 - 8
Hex1: a new human Rad2 nuclease family member with homology to yeast exonuclease 1; Wilson DM 3rd et al.; Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication . We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product . Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair . Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity . Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development . HEX1 gene equivalents were found in all vertebrates examined . The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3645 - 50
Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog; Alleva JL et al.; FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis . A gene with sequence similarity to FEN-1 protein-encoding genes, rad2 +, has been identified in Schizosaccharomyces pombe . We report the overexpression, purification, and character-ization of the putative S.pombe FEN-1 homolog, Rad2p . A GST-Rad2p fusion protein was over-expressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography . Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity . DNA cleavage analysis of several different oligodeoxynucleotide structuresindicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exo-nuclease activities . GST-Rad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DNA . This is the first report on the biochemical characterization of S.pombe Rad2p . The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed.

Micron, 1998 Apr-Jun, 29(2-3), 207 - 33
The ultrastructure of yeast: cell wall structure and formation; Osumi M; Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology . The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of these methods is first presented in this review . The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after thin sectioning and freeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectioning . C . albicans protoplasts are regenerated to synthesize microfibrils on their surface . They are aggregated into thicker bundles which are intermeshed, forming a wide-meshed network of long fibrils . These microfibrillar structures are chains of beta-1,3-glucan which are broken down after treatment with beta-1,3-glucanase . Morphologically identical microfibrils are synthesized in vitro by a cell-free system in which the active cell membrane fraction as a source of beta-1,3-glucan synthetase and UDP glucose as the sole substrate are used . The diameter of an elemental fibril of beta-glucan is estimated to be 2.8 nm from the pattern of autocorrelation of the image obtained by computer processing . In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, resulting in the occurrence of short fibrils . These electron microscopic data suggest that aculeacin A inhibits not only the synthesis of beta-1,3-glucan but the aggregation of microfibrils of this polysaccharide, allowing formation of the crystalline structure . On the basis of the cumulative data obtained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine particles which are then transformed into thin fibrils with single to multiple strands which appear to be oriented parallel to each other so that they develop into fibrillar structures . This process of assembly of beta-glucan molecules leads to the development of a fibrous network within the regenerating Candida cell wall . Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphics, of the regeneration system of Schizosaccharomyces pombe protoplasts: after 10 min of regeneration, the protoplasts begin to grow fibrillar substances of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts . The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick . The microfibrils twist around each other and develop into 8 nm thick fibrils forming flat bundles 16 nm thick . Interfibrillar spaces are gradually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h . Treatment of reverting protoplasts with RuO4 provided clear TEM images of glucan fibrils with high electron density . The relationship between cell wall regeneration and intracellular organelles was examined by using serial thin sections stained with PATAg and computer-aided three-dimensional reconstruction . The secretory vesicles in a protoplast had increased markedly by 1.4, 3.4, and 5.8 times at 1.5, 3.0, and 5 h, respectively . Three-dimensional analysis indicates that Golgi apparatuses are located close together in the nucleus of the protoplast and are dispersed into the cytoplasm during the progress of cell wall formation.

J Bacteriol, 1998 Aug, 180(15), 3992 - 6
Multicellular stalk-like structures in Saccharomyces cerevisiae; Engelberg D et al.; Stalk formation is a novel pattern of multicellular organization . Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures . We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers . Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures . We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces . We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms . Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.

Plant J, 1998 Apr, 14(1), 75 - 81
Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue; Taylor RM et al.; A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach . The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pi) of 8.20 . Alignment of the A . thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42-45% identity, 61-66% similarity), particularly around the active site . Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species . Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S . cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase . The level of the DNA ligase transcript was not increased in A . thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation . However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells.

J Cell Biol, 1998 Jul 27, 142(2), 457 - 71
Regulation of cell polarity by microtubules in fission yeast; Sawin KE et al.; To investigate the role of microtubules in regulating cell polarity in Schizosaccharomyces pombe, we have developed a system in which normally cylindrical fission yeast synchronously form branched cells at high frequency upon treatment with the microtubule-depolymerizing drug thiabendazole (TBZ) . Branching depends on both elevated temperature and cell cycle state and occurs at high frequency only when TBZ is added to cells that have not yet passed through New-End Take-Off (NETO), the normal transition from monopolar to bipolar growth . This suggests that microtubules may be of greatest physiological importance for the maintenance of cell shape at specific points in the cell cycle . The localization of three different proteins normally found at cell ends-cortical F-actin, tea1, and an ral3 (scd2)-green fluorescent protein (GFP) fusion-is disrupted by TBZ treatment . However, these proteins can eventually return to cell ends in the absence of microtubules, indicating that although their localization to ends normally depends on microtubules, they may recover by alternative mechanisms . In addition, TBZ induces a shift in ral3-GFP distribution from cell ends to the cell middle, suggesting that a protein complex containing ral3 may be part of the cue that specifies the position of branch formation.

J Biol Chem, 1998 Jul 31, 273(31), 19747 - 55
Characterization of the two small subunits of Saccharomyces cerevisiae DNA polymerase delta; Gerik KJ et al.; Yeast DNA polymerase delta (Poldelta) has three subunits of 125, 58, and 55 kDa . The gene for the 125-kDa catalytic subunit (POL3) has been known for several years . Here we describe the cloning of the genes for the 58- and 55-kDa subunits using peptide sequence analysis and searching of the yeast genome data base . The 58-kDa subunit, encoded by the POL31 gene, shows 23-28% sequence similarity to the 48-kDa subunit of human Poldelta and to S . pombe Cdc1 . POL31 is allelic to HYS2 and SDP5 . The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base) . Very limited sequence similarity was observed between Pol32p and Schizosaccharomyces pombe Cdc27, the functionally analogous subunit in S . pombe Poldelta . The POL32 gene is not essential, but a deletion mutant shows cold sensitivity for growth and is sensitive to hydroxyurea and DNA damaging agents . In addition, lethality was observed when the POL32 deletion mutation was combined with conditional mutations in either the POL3 or POL31 gene . Pol32Delta strains are weak antimutators and are defective for damage-induced mutagenesis . The POL32 gene product binds proliferating cell nuclear antigen . A gel filtration analysis showed that Pol32p is a dimer in solution . When POL31 and POL32 were co-expressed in Escherichia coli, a tetrameric (Pol31p.Pol32p)2 species was detected by gel filtration, indicating that the two subunits form a complex.

Yeast, 1998 Jun 15, 14(8), 747 - 57
Functional analysis of the Saccharomyces cerevisiae UBC11 gene; Townsley FM et al.; UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe . We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction . Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from delta ubc11 or delta ubc4 delta ubc11 cells . These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S . cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C.

Yeast, 1998 Jun 15, 14(8), 733 - 46
The C-terminal hydrophobic repeat of Schizosaccharomyces pombe heat shock factor is not required for heat-induced DNA-binding; Saltsman KA et al.; The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain . Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S . pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR . We examined the role that the CTR plays in the regulation of S . pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf . Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding . Deletion of the CTR did, however, slightly lower the temperature required for maximal activation . In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature . Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock . These experiments demonstrate that appropriate regulation of the DNA binding activity of S . pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf.

Yeast, 1998 Jun 15, 14(8), 701 - 10
Pombe: a gene-finding and exon-intron structure prediction system for fission yeast; Chen T et al.; A special program developed by the authors, called Pombe, identifies protein coding regions in the Schizosaccharomyces pombe genome . Linear discriminant analysis was applied to predict 5'-terminal, internal, 3'-terminal exons (coding-exon) and introns . The accuracy of the prediction was tested by cross verifications . The sensitivity, specificity and correlation coefficient for the internal exon prediction were 98.5%, 99.9% and 98.3% respectively at the nucleotide level . Open reading frames were studied and used to predict intron-less genes: 99.0% of such genes were identified with correct stopping sites . The gene structure was determined by dynamic programming and the prediction achieved 97.0% correlation coefficient at the nucleotide level . The program is available at http:(/)/clio.cshl.org/genefinder.

Mol Cell Biol, 1998 Aug, 18(8), 4488 - 98
An RNA binding protein negatively controlling differentiation in fission yeast; Tsukahara K et al.; The fission yeast Schizosaccharomyces pombe starts sexual development when starved for nutrients and simultaneously activated by mating pheromones . We have identified a new gene regulating the onset of this process . This gene, called nrd1(+), encodes a typical RNA binding protein that preferentially binds poly(U) . Deletion of nrd1(+) causes cells to initiate sexual development without nutrient starvation . We have found that the biological role of nrd1(+) is to block the onset of sexual development by repressing the Ste11-regulated genes essential for conjugation and meiosis until cells reach a critical level of starvation.

Mol Gen Genet, 1998 Jun, 258(6), 663 - 70
Global control of meiotic recombination genes by Schizosaccharomyces pombe rec16 (rep1); Ding R et al.; The Schizosaccharomyces pombe rec16-125 mutation reduces meiotic recombination, delays premeiotic DNA synthesis, and reduces the accumulation of some but not other rec gene transcripts . To elucidate the function of the Rec16 global meiotic regulator, we cloned and sequenced rec16 . The data revealed that rec16 is identical to rep1, which was previously shown to encode a protein with a zinc-finger motif required for pre-meiotic DNA synthesis . Transcripts of rec16 (rep1) were strongly induced and subsequently degraded during meiosis . In a rec16 (rep1) deletion mutant, meiotic induction of the seven rec genes tested, which appear to be directly involved in meiotic recombination, was significantly reduced or essentially abolished . Deletion of 80% of the gene essentially abolished meiotic recombination, whereas strains deleted for approximately one-quarter of the gene, from either end, retained partial activity . The rec16-125 mutation strongly reduced recombination in the intervals tested on chromosomes I and III, a phenotype characteristic of mutations in rec genes, such as rec7, whose expression requires Rec16 (Rep1) . These results show that Rec16 (Rep1) does not have the regional specificity of Rec10 . We infer that Rec16 (Rep1) is a transcriptional activator that is required for meiotic replication and recombination because it plays a role in the transcriptional induction of the rec and other meiosis-specific genes.

Biochem Cell Biol, 1998, 76(1), 107 - 13
Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe; Tsai CS et al.; Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pmbe have been purified 250-fold by an identical three-step . Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase . Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S . pombe (fission yeast) . Hexokinase promotes Mg(2+)-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex . ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose . The Mg2+ activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group . G6PDH catalyzes NADP(+)-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+ as the leading reactant . High NADP+ concentration inhibits the dehydrogenase by forming the dead-end ternary complex . In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP . Thus, the oxidative pentose phosphate pathway in S . pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.

J Biol Chem, 1998 Jul 17, 273(29), 18490 - 8
Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast; Sells MA et al.; p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac . Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined . We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity . Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe . Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family . Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S . pombe . Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity . A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains . These results show that S . pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.

J Biol Chem, 1998 Jul 17, 273(29), 18340 - 6
Identification of a human homologue of the Schizosaccharomyces pombe rad17+ checkpoint gene; Parker AE et al.; In the fission yeast Schizosaccharomyces pombe the rad17+ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints . We have identified a human cDNA homologue of the S . pombe rad17+ checkpoint gene, designated Hrad17 . Hrad17 has 49% identity to the S . pombe rad17+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level . Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines . Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3-21.2 . This region is subject to loss of heterozygosity in several human cancers . To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17 . We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.

J Biol Chem, 1998 Jul 17, 273(29), 18332 - 9
A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease; Parker AE et al.; In the fission yeast Schizosaccharomyces pombe the rad1(+) gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints . We have identified a human homologue of the S . pombe rad1(+) gene, designated Hrad1, as well as a mouse homologue: Mrad1 . Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B . Hrad1A has 60% identity to the S . pombe rad1+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level . Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines . Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13 . 2-13.3 . This region is subject to loss of heterozygosity in several human cancers . Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins . REC1 has previously been characterized as a 3' --> 5' exonuclease with a C-terminal domain essential for cell cycle checkpoint function . We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3' --> 5' exonuclease activity, whereas HisHrad1B lacks such activity . The biological functions of the two proteins remain to be determined.

Mol Pharmacol, 1998 Jul, 54(1), 213 - 9
Sensitivity to cisplatin and platinum-containing compounds of Schizosaccharomyces pombe rad mutants; Perego P et al.; The role of genes that affect response to radiation in determining sensitivity to platinum-containing compounds was studied using a panel of 23 strains of the yeast Schizosaccharomyces pombe . The radiation-hypersensitive mutants all had the same genetic background and most of them contained mutations that disabled either cell cycle checkpoints or DNA repair . The tested platinum compounds included cisplatin and two complexes containing diaminocyclohexane (oxaliplatin and tetraplatin), two ammine/cyclohexylamine complexes with different orientation of the leaving groups (JM216 and JM335) and a multinuclear platinum complex (BBR 3464) . The cytotoxic effect of the selected platinum complexes was evaluated by using a microtiter growth inhibition assay with a 48 hr exposure to drug . The mutants fell into three groups with respect to sensitivity to cisplatin: four mutants (rad2, -7, -11, -15) exhibited minimal change in sensitivity; fifteen mutants (rad4-6, -8-10, -12-14, -16-17, -19-21, and -22) were 5.1-21.7-fold hypersensitive; only rad1 and -3 mutants, defective in checkpoints, and rad18, defective in repair, displayed a marked hypersensitivity . None of the mutants demonstrated appreciable change in sensitivity to JM216 presumably as a consequence of a lack of resistance of the wild-type strain, whereas a moderate increase in sensitivity to JM335 was observed for most of the mutants, and hypersensitivity to BBR3464 was observed only in rad1 and -3 . No relevant changes in sensitivity to tetraplatin were observed . Most of the mutants, with the exception of rad2, -7, and -15, were hypersensitive to oxaliplatin . These findings demonstrate that specific mutations have disparate effects on the profile of sensitivity to different members of the same class of cytotoxic agents, which provides genetic evidence that different mechanisms are involved in differential cytotoxicity induced by Pt compounds . The results also demonstrate the utility of such a panel of mutants, constructed on the same genetic background, for detecting specific cellular response; presumably, this reflects the recognition or processing of specific DNA adducts . In conclusion, because the rad1 and rad3 gene products are determinants of cellular response to a large number of platinum-containing compounds, the present results support a critical role of genes involved in cell cycle control in cellular sensitivity to these agents.

J Bacteriol, 1998 Jul, 180(14), 3727 - 9
A minisatellite sequence within the propeptide region of the vacuolar carboxypeptidase Y gene of Schizosaccharomyces pombe; Ingavale SS et al.; We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast Schizosaccharomyces pombe . The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene . The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of S . pombe (S . pombe var . pombe and S . pombe var . malidevorans) . The minisatellite sequence, however, appeared to be species specific and was absent in other members of the Schizosaccharomyces genus . This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.

Mol Cell Biochem, 1998 Jun, 183(1-2), 125 - 32
Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli; Dibrov P et al.; Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe . It is vitally important for sodium export and pH homeostasis in this organism . Recently, the sod2 gene has been cloned and sequenced . However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed . In the present work we examined physiological consequences of expression of sod2 in E . coli . To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E . coli strain MH1 which has impaired sodium exchange . The effect of sod2 expression on E . coli varied depending on the E . coli genotype . When sod2 was expressed in BL21 cells which have normal Na+/H+ antiporters, the result was a Li+ sensitive phenotype . LiCl completely arrested or prevented growth of BL21 E . coli transformed with the sod2 gene . The effect on growth was pronounced in media of low external pH . Sod2 was then expressed in E . coli MH1 which is devoid of endogenous Na+/H+ antiporters . These cells became more resistant to external LiCl, but only in Na+ containing media . In the absence of external Na+, the presence of sod2 reduced growth . The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na+/H+ antiporter in conjunction with different housekeeping systems of E . coli host cells.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8159 - 64
sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast; Jallepalli PV et al.; In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein . Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast . In this paper we describe one such component, encoded by the sud1(+) gene . sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast . Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators . Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis . Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo . Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites . These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1 . Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.

Gene, 1998 Jul 3, 214(1-2), 131 - 7
Identification of a novel casein kinase-1 homolog in fission yeast Schizosaccharomyces pombe; Kitamura K et al.; Fission yeast cells lacking either the ste9+- or rum1+ function cannot enter the cell differentiation pathway upon nutritional starvation . Sterility in both mutants is suppressed by the srs1-S41 mutation . A gene encoding a novel casein kinase-1 (CK1) isoform, cki3+, was isolated as a high-copy-number suppressor gene of the srs1 mutation . Cki3 protein is structurally more related to the Cki/Yck subfamily proteins than those of the Hhp/Hrr25 subfamily . A mutant cki3 gene in which a highly conserved lysine residue in the kinase subdomain II was substituted to arginine lost the ability to recover the growth defect in the srs1 mutant, indicating that catalytic activity was necessary for suppression . Gene disruption revealed that cki3+ was dispensable for cell viability, and cells lacking functional cki3+ exhibited no characteristic phenotype . Thus, S . pombe has three highly related CK1 isoforms (Cki1, Cki2 and Cki3), but none of them has an essential function.

Gene, 1998 Jul 3, 214(1-2), 101 - 12
Mosaic structure of the cox2 gene in the petite negative yeast Schizosaccharomyces pombe: a group II intron is inserted at the same location as the otherwise unrelated group II introns in the mitochondria of higher plants; Schafer B et al.; In contrast to homologous genes in other fungal mitochondrial genomes, the gene encoding subunit 2 of cytochrome oxidase (cox2) in several Schizosaccharomyces pombe strains contains a large group II intron . Its 2436 nucleotides can be folded into a typical group II intron secondary structure, possessing all the expected sequence motifs for subgroup IIA1 (Michel et al., 1989) . This intron is remarkable for the following reasons: (i) Five nucleotide changes were observed compared with the continuous form of the cox2 gene in the reference strain 50 at the 3'-exon sequence, but not in the 5'-exon . (ii) One of these changes occurred at the splice point leading to a serine instead of a threonine residue in the deduced cox2 polypeptide . In all cases, the alterations resulted in the replacement of more frequently used codons by rare ones . (iii) Although the intron is able to undergo splicing, the sequence motifs thought to be necessary for interaction between the 5'-exon and the intron during the splicing process (the EBS1/IBS1 as well as the EBS2/IBS2 pairings) are unusual . (iv) The intron is inserted at the same location in the cox2 gene as the otherwise unrelated intron from higher plants.

Nucleic Acids Res, 1998 Jul 15, 26(14), 3319 - 22
Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases; Gross T et al.; We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins . All contain the Homol D-box in their promoter . We have shown that Homol D is, in this promoter type, the TATA-analogue . Many promoters contain the Homol E-box, which serves as a proximal activation sequence . Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28 . The budding yeast Saccharomyces cerevisiae has no L28 equivalent . Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes . Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.

Genetics, 1998 Jul, 149(3), 1265 - 75
Isolation and characterization of new fission yeast cytokinesis mutants; Balasubramanian MK et al.; Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring . To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel . Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring . Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized . Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118 . The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively . The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p . In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.

Genetics, 1998 Jul, 149(3), 1251 - 64
A screen for genes involved in the anaphase proteolytic pathway identifies tsm1(+), a novel Schizosaccharomyces pombe gene important for microtubule integrity; Grishchuk EL et al.; The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK) . Because nuc2(+) encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S . pombe mutants defective in the anaphase-promoting pathway . In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains . Two of these are alleles of cut1(+), but tsm1-512 maps to a novel genetic location . The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle . After shift of early G2 cells to 36 degrees, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation . The chromosomes, however, often fail to segregate properly . Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.

Genetics, 1998 Jul, 149(3), 1221 - 33
Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity; Gould KL et al.; A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast . We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1 . The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD . A cdc24 null mutant was generated by homologous recombination . Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene . The transcript of cdc24(+) is present at constant levels throughout the cell cycle . Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase . Arrest is accompanied by a rapid loss of viability and chromosome breakage . An S . pombe homolog of the replicative DNA helicase DNA2 of S . cerevisiae suppresses cdc24 . These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

Genetics, 1998 Jul, 149(3), 1191 - 204
The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements; Virgin JB et al.; Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination) . Recombination hotspots are important elements in controlling meiotic allelic recombination . We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination . Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S . pombe . The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination . Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26 . These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Genes Dev, 1998 Jul 1, 12(13), 1986 - 97
Identification of Xenopus SMC protein complexes required for sister chromatid cohesion; Losada A et al.; The structural maintenance of chromosomes (SMC) family is a growing family of chromosomal ATPases . The founding class of SMC protein complexes, condensins, plays a central role in mitotic chromosome condensation . We report here a new class of SMC protein complexes containing XSMC1 and XSMC3, Xenopus homologs of yeast Smc1p and Smc3p, respectively . The protein complexes (termed cohesins) exist as two major forms with sedimentation coefficients of 9S and 14S . 9S cohesin is a heterodimer of XSMC1 and XSMC3, whereas 14S cohesin contains three additional subunits . One of them has been identified as a Xenopus homolog of the Schizosaccharomyces pombe Rad21p implicated in DNA repair and the Saccharomyces cerevisiae Scc1p/Mcd1p implicated in sister chromatid cohesion . 14S cohesin binds to interphase chromatin independently of DNA replication and dissociates from it at the onset of mitosis . Immunodepletion of cohesins during interphase causes defects in sister chromatid cohesion in subsequent mitosis, whereas condensation is unaffected . These results suggest that proper assembly of mitotic chromosomes is regulated by two distinct classes of SMC protein complexes, cohesins and condensins.

Mol Gen Genet, 1998 May, 258(4), 389 - 96
Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe; Neu R et al.; In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe . In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome . Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus . As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene . The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol . The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene . Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae . We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype . The significant similarity of the urf a gene to the mitochondrial var1 gene of S . cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

Mol Gen Genet, 1998 May, 258(3), 279 - 87
Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe; Sugaya K et al.; A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4 . To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpbl gene, which encodes RpII LS . Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures . First, we examined the effects of the mutation on haploid yeast cells . The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature . When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed . Tetrad analysis suggested that these phenotypes were associated with the mutation . In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene . An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis . The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability . These observations demonstrate that the S . pombe rpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.

Curr Genet, 1998 Jun, 33(6), 445 - 50
Isolation of a Trichoderma reesei cDNA encoding GTP: a-D-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation; Kruszewska JS et al.; A cDNA coding for GTP: alpha-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase . The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids . Sequence comparisons demonstrate 70% identity with the S . cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue . No similarity was found with the MPD synthase encoded by the S . cerevisiae DPM1 gene . The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S . cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores . Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S . cerevisiae dpm1-6 mutant.

Mol Microbiol, 1998 May, 28(4), 729 - 41
Schizosaccharomyces pombe isp4 encodes a transporter representing a novel family of oligopeptide transporters; Lubkowitz MA et al.; We have recently cloned an oligopeptide transport gene from Candida albicans denoted OPT1 . This gene showed significant sequence similarity to three open reading frames (ORFs) with no previously established function: isp4 from Schizosaccharomyces pombe and Saccharomyces cerevisiae YJL212C and YPR194C, identified during the genome project . The S . pombe gene isp4 was originally identified by Sato et al . as a gene that was upregulated through nitrogen starvation induction of meiosis . However, an isp4delta strain exhibited a wild-type phenotype with respect to sexual differentiation . We have found that the same isp4delta strain is deficient in tetrapeptide transport activity as measured by its resistance to toxic tetrapeptides, by its inability to accumulate a radiolabelled tetrapeptide and by the inability to use tetrapeptides as a sole source of an amino acid to satisfy an auxotrophic requirement . Similarly, we found that the ORF YPR194C from S . cerevisiae encodes an oligopeptide transporter . Sequence analyses as well as physiological evidence has led us to propose that the proteins encoded by isp4 and the genes identified from S . cerevisiae and C . albicans comprise a new group of transporters specific for small oligopeptides, which we have named the OPT family.

J Mol Biol, 1998 Jun 19, 279(4), 703 - 12
Two large subunits of the fission yeast RNA polymerase II provide platforms for the assembly of small subunits; Ishiguro A et al.; The subunit-subunit contact network was analyzed for the Schizosaccharomyces pombe RNA polymerase II consisting of ten putative subunits . Previously we carried out far-Western blot analysis of bimolecular interaction with radio-labeled subunit 3 and 5 probes . Here we extended the analysis using another six small-sized subunits as probes . Taking the results together the subunit-subunit interaction was observed for a total 18 (or 19) combinations . All eight small-sized subunits exhibited binding activities to two large subunits, Rpb1 and Rpb2 . In addition, bimolecular interaction was observed for the combinations of Rpb3-Rpb5, Rpb3-Rpb11 (and Rpb5-Rpb8/11) . The subunit-subunit contact within the assembled RNA polymerase was then analyzed by protein-protein cross-linking using five species of bifunctional cross-linkers with different length and specificity . Cross-linking was observed for a total of 19 combinations, including five combinations between small subunits, Rpb3-Rpb10, Rpb3-Rpb11, Rpb5-Rpb6, Rpb6-Rpb7 and Rpb6-Rpb8 . The results altogether indicate that two large subunits Rpb1 and Rpb2 provide the platform for assembly of small subunits and also small subunits interact with each other for limited combinations . Direct contact of the two large subunits, Rpb1 and Rpb2, was also demonstrated by cross-linking .

EMBO J, 1998 Jun 1, 17(11), 3066 - 77
Nascent transcription from the nmt1 and nmt2 genes of Schizosaccharomyces pombe overlaps neighbouring genes; Hansen K et al.; We have determined the extent of the primary transcription unit for the two highly expressed genes nmt1 and nmt2 of Schizosaccharomyces pombe . Transcription run-on analysis in permeabilized yeast cells was employed to map polymerase density across the 3'-flanking region of these two genes . Surprisingly, polymerases were detected 4.3 kb beyond the nmt1 polyadenylation {poly(A)} site and 2.4 kb beyond the nmt2 poly(A) site, which in each case have transcribed through an entire convergent downstream transcription unit . However, the steady-state levels of both downstream genes were unaffected by the high level of nmt1 or nmt2 nascent transcription . Analysis of nmt1 and nmt2 RNA 3' end formation signals indicates that efficient termination of transcription requires not only a poly(A) signal but also additional pause elements . The absence of such pause elements close to the poly(A) sites of these genes may account for their extended nascent transcripts.

Mol Cell Biol, 1998 Jul, 18(7), 4141 - 8
Functional conservation of the transportin nuclear import pathway in divergent organisms; Siomi MC et al.; Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins {hnRNP}) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm . hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro . Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned . To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it . In an exhaustive screen of the S . cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p . We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif . NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells . Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro . We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs . Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.

Mol Cell Biol, 1998 Jul, 18(7), 4097 - 108
Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes; Ohi R et al.; Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression . To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens . Supporting the notion that these Cdc5 gene family members are functionally homologous to S . pombe cdc5(+), human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S . pombe cdc5-120 mutant . Furthermore, S . cerevisiae CEF1 (S . cerevisiae homolog of cdc5(+)), like S . pombe cdc5(+), is essential during G2/M . The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo . However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo . Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity . Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5 . Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression.

Mol Cell Biol, 1998 Jul, 18(7), 3782 - 7
Tyrosine phosphorylation of cdc2 is required for the replication checkpoint in Schizosaccharomyces pombe; Rhind N et al.; The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea . In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15 . On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism . We have undertaken biochemical and genetic studies to resolve this controversy . We report that the DNA replication checkpoint in S . pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2 . Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis . These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.

Curr Biol, 1998 May 21, 8(11), 633 - 41
Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis; Kumada K et al.; BACKGROUND: The Cut1 and Cut2 proteins of the fission yeast Schizosaccharomyces pombe form a complex and are required for the separation of sister chromatids during anaphase . Polyubiquitinated Cut2 degrades at the onset of anaphase and this degradation, like that of mitotic cyclin, is dependent on the anaphase-promoting complex/cyclosome . Expression of Cut2 that cannot be degraded blocks sister chromatid separation and anaphase spindle elongation . Here, we have investigated the role of the Cut1-Cut2 interaction in sister chromatid separation . RESULTS: The carboxyl terminus of Cut2 interacts with the amino terminus of Cut1, and temperature-sensitive Cut2 mutants expressed Cut2 proteins that contain substitutions in the carboxyl terminus and fail to interact with Cut1, resulting in aberrant anaphase . Localization of Cut1 alters dramatically during the cell cycle . Cut1 is retained in the cytoplasm during interphase and moves to the mitotic spindle pole bodies and the spindle upon entry into prophase, when spindles are formed . The association between Cut2 and Cut1 is needed for the localization of Cut1 to the spindles, as Cut1 remains unbound to the spindle if complex formation is impaired . Cut2 degrades during anaphase, but Cut1 remains bound to the anaphase spindle . This association with the anaphase spindle requires the conserved carboxyl terminus of Cut1 . CONCLUSIONS: Complex formation between Cut1 and Cut2 is needed for the onset of normal anaphase . Cut2 is required for loading Cut1 onto the spindle at prophase and Cut2 proteolysis is needed for the active participation of Cut1 in sister chromatid separation.

Curr Biol, 1998 May 21, 8(11), 611 - 21
Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body; Eng K et al.; BACKGROUND: An actomyosin-based contractile ring plays a pivotal role in cytokinesis . Despite the identification of many components of the ring, the steps involved in its assembly are unknown . The fission yeast Schizosaccharomyces pombe is an attractive organism in which to study cytokinesis because its cell cycle has been well characterized; it divides by medial fission using an actomyosin ring; and a number of S . pombe mutants defective in actomyosin ring assembly have been isolated . Here, we have characterized one such mutant, rng2 . RESULTS: Temperature-sensitive rng2 mutants accumulated F-actin cables in the medial region of the cell but failed to organize the cables into a ring . In rng2-null mutants, only a spot-like structure containing F-actin was detected . The rng2+ gene encodes a protein related to human IQGAP1, a protein that binds actin and calmodulin and is a potential effector for the Rho family of GTPases . Rng2p localized to the actomyosin ring and to the spindle pole body (SPB) of interphase and mitotic cells . Localization of Rng2p to the actomyosin ring but not the SPB required F-actin . Rng2p interacted with calmodulin, a component of the SPB and the actomyosin ring . The rng2 gene showed genetic interactions with three other actomyosin ring assembly mutants, cdc4, cdc12, and rng5 . CONCLUSIONS: The S . pombe IQGAP-related protein Rng2p is a component of the actomyosin ring and the SPB and is required for actomyosin ring construction following assembly of F-actin at the division site.

Yakugaku Zasshi, 1998 Apr, 118(4), 112 - 26
Natural organic compounds that affect to microtubule functions; Iwasaki S; Microtubules (MT), composed of a protein tubulin (TN) alpha,beta-heterodimer with concomitant other proteins, microtubule associated proteins (MAPs and tau), are known to be the main component of spindles in a mitotic apparatus of eucaryotic cells, and are also involved in many other basic and essential cell functions . There are a number of natural and synthetic compounds that interfere with MT function to cause the mitotic arrest of eucaryotic cells . Such antimitotic agents show a broad biological activity, and can be used for medicinal and agrochemical purposes . On the other hand, they are also important as the biochemical tools for understanding the dynamics of MT network . Most of such antimitotic agents, with a few exceptions, bind to beta-TN . Among them, colchicine (CLC), vinblastine (VLB) and taxol have been of major importance in biochemical studies of MT and in studies of their intracellular functions . The former two both inhibit MT assembly but their binding sites on beta-TN are different; CLC-site and VLB-site, and many MT inhibitors bind to either sites . Taxol bind to TN at a site other than CLC-site and VLB-site, and promote MT assembly . We have worked on a variety of antimitotic agents that bind to CLC, VLB or taxol-site, in discoveries, structures, biological actions and/or interactions with TN . In this paper, I summarized the results of our studies on VLB-site ligands; (1) rhizoxin (RZX), isolated as a phytotoxin produced by a plant pathogenic fungus, and its related compounds, (2) derivatives of ansamitocin P-3 (ASMP3) (maytansinoid: MAY), isolated as a cytotoxic metabolite of an Actinomycete, (3) phomopsin A (PMSA), isolated as a mycotoxin produced by a plant parasitic fungus, (4) dolastatin 10 (DLS10), isolated as a cytotoxic metabolite of a see animal, (5) ustiloxins (USL) A-F, isolated as a mycotoxin produced by a plant pathogenic fungus, (6) arenastation A (ARSA), isolated as a cytotoxic metabolite of a sponge, and its synthetic analogs . From our studies on interactions of these VLB-site ligands with TN, we showed that the presence of a distinct RZX/MAY-binding site which only partially overlap with VLB-site, and that PMSA, DLS10, USLs and ARSA bind to the RZX/MAY site . RZX, ASMP3 and ARSA inhibit the growth of a variety of fungi, including Aspergillus nidulans . In order to obtain information as to the drug-TN interaction at the RZX/MAY site, RZX-resistant beta-TN gene mutants were isolated from RZX-sensitive wild-type A . nidulans . In all the beta-TN gene mutants, single amino acid (100th) alteration, asparagine-to-isoleucine, was observed . Sequence displacement experiments confirmed that this alteration conferred resistance to RZX and ASMP3, and also to ARSA . This resistance mechanism was further verified with yeasts Schizosaccharomyces pombe and Saccharomyces serevisiae . All the natural ligands mentioned above show potent cytotoxicity against human and murine tumor cells, but VLB, PMSA, DLS10 and USLA are inactive to both RZX-sensitive and -resistant fungal strains.

Nat Biotechnol, 1996 May, 14(5), 600 - 5
A functional screen in yeast for regulators and antagonizers of heterologous protein tyrosine kinases; Superti-Furga G et al.; Tyrosine phosphorylation exerts a pivotal role in cell regulation processes of higher eukaryotes . Tight control of the activity of protein tyrosine kinases is crucial for ordered phosphorylation to occur . We have developed a functional screen for tyrosine kinase regulators using c-Src, the first cellular protein tyrosine kinase described, as a prototype; and fission yeast, Schizosaccharomyces pombe, as a genetically amenable host system . Inducible expression of c-Src in fission yeast is lethal . We have screened human cDNA libraries for clones able to counteract the lethal effect of Src . Two different classes of cDNAs, which we called SAS for sequences antagonizing Src, were obtained . The first class encodes for the protein tyrosine kinase Csk, known to regulate Src activity through phosphorylation of the C-terminal tyrosine . The second class consists of clones encoding three different tyrosine phosphatases, counteracting Src action by dephosphorylation of Src substrates and by dephosphorylation of Src itself . The system described here can be applied to identify regulators of other heterologous tyrosine kinases, including receptor-type tyrosine kinases, which impair growth of S . pombe.

Biochemistry, 1998 Jun 9, 37(23), 8282 - 8
Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast; Dibrov P et al.; We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe . We mutated all eight His residues of sod2 into Arg . Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S . pombe in LiCl-containing medium . Mutant S . pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium . When His367 was replaced by Asp, sodium export of S . pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type . We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers . S . pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium . In addition, sodium-dependent proton influx at external pH 6 . 1 was impaired . Sodium export from S . pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export . The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.

J Biochem (Tokyo), 1998 May, 123(5), 883 - 90
Human dis3p, which binds to either GTP- or GDP-Ran, complements Saccharomyces cerevisiae dis3; Shiomi T et al.; Saccharomyces cerevisiae Dis3p, which interacts with Ran/Gsp1p, complements Schizosaccharomyces pombe dis3-54 . Consistent with the functional conservation of Dis3p in S . cerevisiae and S . pombe, the human ORF (accession number: R27667) was found to be highly homologous to yeast Dis3p . Based on its nucleotide sequence, we cloned a full-sized human DIS3 cDNA . The cloned human cDNA partly but significantly restored the temperature-sensitivity of S . cerevisiae dis3 . Thus, Dis3p was found to be structurally and functionally conserved from yeast to mammals . Consistent with the report that S . cerevisiae Dis3p is identical to Rrp44p, which comprises the exosome involved in ribosomal RNA processing, S . cerevisiae Dis3p was found to be localized in the nucleolus . Similar to S . cerevisiae Dis3p, human Dis3p enhanced RCC1-stimulated nucleotide release from Ran, in a dose-dependent manner, and bound to GTP- or GDP-Ran.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3247 - 54
Genetic characterisation of hda1+, a putative fission yeast histone deacetylase gene; Olsson TG et al.; hda1+ (histone deacetylase 1) is a fission yeast gene which is highly similar in sequence to known histone deacetylase genes in humans and budding yeast . We have investigated if this putative histone deacetylase contributes to transcriptional silencing in the fission yeast Schizosaccharomyces pombe . A precise deletion allele of the hda1+ open reading frame was created . Cells lacking the hda1+ gene are viable . However, genetic analysis reveals that cells without hda1 + display enhanced gene repression/silencing of marker genes, residing adjacent to telomeres, close to the silent mating-type loci and within centromere I . This phenotype is very similar to that recently reported for rpd3 mutants both in Drosophila and budding yeast . No defects in chromosome segregation or changes in telomere length were detected . Cells lacking the hda1+ gene display reduced sporulation . Growth of hda1 cells is partially inhibited by low concentrations of Trichostatin A (TSA), a known inhibitor of histone deacetylase enzymes . TSA treatment is also able to overcome the enhanced silencing found in heterochromatic regions of hda1 cells . These results indicate a genetic redundancy with respect to deacetylase genes and partially overlapping functions of these in fission yeast . The significance of these results is discussed in the light of recent discoveries from other eukaryotes.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3077 - 83
Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair; Qiu J et al.; Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair . An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light . Deletion of the RAD2 gene in S . cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains . In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease . Expression of the EXO1 gene is UV inducible . Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants . Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA . The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants . This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER . Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.

Biol Chem, 1998 Apr-May, 379(4-5), 591 - 4
Substitution of the conserved phenylalanine in the S-adenosyl-L-methionine binding site of M.MspI with tyrosine modifies the kinetic properties of the enzyme; Pinarbasi E et al.; Cytosine (C-5)-specific DNA methyltransferases share a set of ten conserved motifs distributed evenly throughout the entire polypeptide chain . The first conserved motif contains a Phe, which is intimately associated with cofactor recognition . In the pseudo-DNA methyltransferase M.SpoI, encoded by the pmt1 gene in Schizosaccharomyces pombe, a Tyr replaces this Phe residue . We describe the properties of a mutant form of M.MspI, a typical cytosine (C-5)-specific DNA methyltransferase, in which Tyr replaces the conserved Phe . This mutant shows differences in ternary complex formation and in the pattern of covalent complex formation with an inhibitory, fluorinated DNA duplex which may be due to anomalous hydrogen bonding between the mutant Tyr hydroxyl group and the catalytic loop of the enzyme or through interference with cofactor binding.

Mol Biol Cell, 1998 Jun, 9(6), 1577 - 88
Functional domains of rep2, a transcriptional activator subunit for Res2-Cdc10, controlling the cell cycle "start"; Tahara S et al.; In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules . This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression . The rep2(+) gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2-Cdc10 complex . To identify the rep2(+) function and molecularly define its domain organization, we reconstituted the Res2-Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae . Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2-Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively . The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

J Biol Chem, 1998 May 8, 273(19), 11456 - 62
Alteration of substrate affinities and specificities of the Chlorella Hexose/H+ symporters by mutations and construction of chimeras; Will A et al.; The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe . By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated . The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI . Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively . An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants . These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway . Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose . Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group . Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein . A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop . The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose . The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.

J Cell Biol, 1998 Jun 1, 141(5), 1217 - 28
FH3, a domain found in formins, targets the fission yeast formin Fus1 to the projection tip during conjugation; Petersen J et al.; Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2 . We describe a new formin homology region, FH3 . FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia . The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs . We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain . Deletion of any FH domain essentially abolished mating . FH3, but neither FH1 nor FH2, was required for Fus1 localization . An FH3 domain-GFP fusion protein localized to the projection tips of mating pairs . Thus, the FH3 domain alone can direct protein localization . The FH3 domains of both Fus1 and the S . pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population . Expression of both FH3-GFP fusions also affected cytokinesis . Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain . Together these data suggest that proteins at multiple sites can interact with FH3 domains.

Mol Microbiol, 1998 Apr, 28(2), 355 - 70
Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans; Cazelle B et al.; The prnA gene codes for a transcriptional activator that mediates proline induction of four other genes involved in proline utilization as a nitrogen and/or carbon source in Aspergillus nidulans . In this paper, we present the genomic and cDNA sequence and the transcript map of prnA . The PrnA protein belongs to the Zn binuclear cluster family of transcriptional activators . The gene shows a striking intron-exon organization, with the putative nuclear localization sequence and the Zn cluster domain in discrete exons . Although the protein sequence presents some interesting similarities with the isofunctional protein of Saccharomyces cerevisiae Put3p, a higher degree of similarity is found with a functionally unrelated protein Thi1 of Schizosaccharomyces pombe . A number of mutations mapping in the prnA gene were sequenced . This comprises a deletion that results in an almost complete loss of the prnA-specific mRNA, a mutation in the putative nuclear localization signal, a proline to leucine mutation in the second loop of the zinc cluster and a cold-sensitive mutation in the so-called 'central region' . Other complete or partial loss of function mutations map in regions of unknown function . We establish that the transcription of the gene is neither self-regulated nor significantly affected by carbon and/or nitrogen metabolite repression.

Nat Genet, 1998 Jun, 19(2), 192 - 5
The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast; Ivanova AV et al.; Heritable inactivation of specific regions of the genome is a widespread, possibly universal phenomenon for gene regulation in eukaryotes . Self-perpetuating, clonally inherited chromatin structure has been proposed as the explanation for such phenomena as position-effect variegation (PEV) and control of segment determination and differentiation in flies, X-chromosome inactivation and parental imprinting in mammals, gene silencing by paramutation in maize and silencing of the mating-type loci in yeasts . We have now found that the clr4 gene, which is essential for silencing of centromeres and the mating-type loci in Schizosaccharomyces pombe, encodes a protein with high homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila . Like Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins that modulate chromatin structure . Site-directed mutations in the conserved residues of the chromo domain confirm that it is required for proper silencing and directional switching of the mating type, like SET domain . Surprisingly, RNA differential display experiments demonstrated that clr4+ can mediate transcriptional activation of certain other loci . These results show that clr4 plays a critical role in silencing at mating-type loci and centromeres through the organization of repressive chromatin structure and demonstrate a new, activator function for Clr4p.

Genes Cells, 1998 Mar, 3(3), 157 - 66
Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe; Miyake S et al.; BACKGROUND: The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts . Six MCM subtypes have been identified in various species . These MCM proteins are thought to bind each other to make a heteromeric complex . The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication . RESULTS: The suppressor mutant of nda4 was isolated and the mutant gene was named sna41 . The sna41-912 mutant demonstrated the ts phenotype, with an elongated cell shape at the restrictive temperature . Cells with 1C DNA content accumulated 2 h after shifting up to the restrictive temperature . This result suggests that sna41 is also involved in DNA replication . The sna41 genomic clone was isolated by a complementation of the ts phenotype of the mutant strain and was sequenced . The sna41 gene encodes a protein of 638 amino acids, which has low homology with CDC45 in S . cerevisiae . The gene disruption analysis showed that sna41 gene is essential for viability . CONCLUSIONS: The S . pombe sna41 mutation suppresses the nda4-108 mutation . Sna41 is involved in DNA replication and may play some roles in the regulation of DNA replication by the MCM proteins.

J Biol Chem, 1998 May 1, 273(18), 11241 - 7
Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum; Mamoun CB et al.; We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family . The predicted coding region is 920 amino acids long, twice the size of other members of this family . We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class . To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast . Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion . Recombinant P . falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction . Each half alone was also active in recombinant form . Using the two-hybrid system, we have shown that the two halves can interact . Gel filtration assay of P . falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity . This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.

Genes Dev, 1998 May 1, 12(9), 1356 - 70
Pom1p, a fission yeast protein kinase that provides positional information for both polarized growth and cytokinesis; Bahler J et al.; Schizosaccharomyces pombe cells have a well-defined pattern of polarized growth at the cell ends during interphase and divide symmetrically into two equal-sized daughter cells . We identified a gene, pom1, that provides positional information for both growth and division in S . pombe . pom1 mutants form functioning growth zones and division septa but show several abnormalities: (1) After division, cells initiate growth with equal frequencies from either the old or the new end; (2) most cells never switch to bipolar growth but instead grow exclusively at the randomly chosen end; (3) some cells mislocalize their growth axis altogether, leading to the formation of angled and branched cells; and (4) many cells misplace and/or misorient their septa, leading to asymmetric cell division . pom1 encodes a putative protein kinase that is concentrated at the new cell end during interphase, at both cell ends during mitosis, and at the septation site after mitosis . Small amounts of Pom1p are also found at the old cell end during interphase and associated with the actin ring during mitosis . Pom1p localization to the cell ends is independent of actin but requires microtubules and Tea1p . pom1 mutations are synthetically lethal with several other mutations that affect cytokinesis and/or the actin or microtubule cytoskeleton . Thus, Pom1p may position the growth and cytokinesis machineries by interaction with both the actin and microtubule cytoskeletons.

Biochim Biophys Acta, 1998 Apr 29, 1397(2), 146 - 50
The gene for ribosomal protein L7a-1 in Schizosaccharomyces pombe contains an intron after the initiation codon; Marchfelder A et al.; The gene encoding ribosomal protein L7a-1 in the fission yeast Schizosaccharomyces pombe is identified by the similarity of its open reading frame to the respective gene in Saccharomyces cerevisiae . The L7a gene is encoded in two different genomic environments as frequently found for ribosomal protein genes in this organism . One of these genes, L75a-1, is located on chromosome 2 . The two consensus promoter elements homol D and homol E are both identified upstream of the start codon of this gene . The ATG start codon is separated from the main reading frame by an intron of 66 nucleotides .

Microbiology, 1998 May, 144 ( Pt 5), 1319 - 30
Environmentally controlled dimorphic cycle in a fission yeast; Sipiczki M et al.; The fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth . This paper shows that the related Schiz . japonicus var . japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases . On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation . The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium) . The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation . The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes . These environmentally controlled morphological transitions make Schiz . japonicus var . japonicus an attractive model for the investigation of cell polarity and morphogenesis.

Genes Cells, 1998 Feb, 3(2), 99 - 110
DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe; Sugino A et al.; BACKGROUND: DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control . An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon . It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts . RESULTS: We have cloned and sequenced S . pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon) . The predicted amino acid sequence of Pol epsilon is highly homologous to that of S . cerevisiae PolII and human Pol epsilon . Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S . cerevisiae PolII holoenzyme (PolII*) . The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells . Furthermore, the Pol epsilon activity from temperature-sensitive S . pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures . CONCLUSION: These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S . pombe, as is PolII in S . cerevisiae . Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.

J Biochem (Tokyo), 1998 Jun, 123(6), 1048 - 54
Two distinct upstream regions are involved in expression of the catalase gene in Schizosaccharomyces pombe in response to oxidative stress; Nakagawa CW et al.; The DNA region responsible for the induction of the catalase gene of Schizosaccharomyces pombe in response to oxidative stress was determined by constructing a series of deletions in the 5'-flanking region of the gene . Cells having deletion -672 (numbered with the transcription start site as +1) to -111 showed no significant difference in catalase expression from the wild-type cells . Cells having deletion -672 to -89 showed reduced basal expression of the catalase mRNA, but retained the ability of induction in response to oxidative stress . Cells having deletion -672 to -55 completely lost the ability to express the catalase mRNA . These results suggested that two regions, -89 to -55 and -111 to -89, are involved in expression of the catalase gene . The DNA region of -89 to -55 overlapped with the Atf1 binding sequence . The Atf1 is a bZIP transcription factor with an important role in stress response under the control of the Spc1 mitogen activated protein (MAP) kinase . Introduction of the atf1(-) or spc1(-) mutation into the mutant having a deletion in -672 to -89 completely abolished the expression of the catalase mRNA . This result indicated that the Spc1-Atf1 cascade is involved in expression of the catalase gene through the region of -89 to -55 . In mutants spc1(-) and atf1(-), basal expression and induction by hydrogen peroxide of catalase mRNA were observed . These results revealed that not only the Atf1 binding site but also another DNA element independent of the Spc1-Atf1 pathway is involved in the expression of the catalase gene in response to oxidative stress in S . pombe . Proteins that bound specifically to each DNA element existed in the cell extract of the wild-type S . pombe.

J Biol Chem, 1998 Apr 24, 273(17), 10210 - 5
Selective inhibition of Ras interaction with its particular effector by synthetic peptides corresponding to the Ras effector region; Ohnishi M et al.; Ras proteins possess multiple downstream effectors of distinct structures . We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms . Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2 . The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity . The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation . The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase . Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide . These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1834 - 40
A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs; Cerritelli SM et al.; Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif . Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif . A very similar amino acid sequence is present in caulimovirus ORF VI proteins . The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic . Point mutations, insertions and deletions indicated that integrity of the motif is important for binding . However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well . Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence . The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.

Curr Biol, 1998 May 7, 8(10), 607 - 10
The helC gene encodes a putative DEAD-box RNA helicase required for development in Dictyostelium discoideum; Machesky LM et al.; DEAD-box RNA helicases, defined by the sequence Asp-Glu-Ala-Asp (DEAD, in single-letter amino-acid code), regulate RNA unwinding and secondary structure in an ATP-dependent manner in vitro {1} and control mRNA stability and protein translation . Both yeast and mammals have large families of DEAD-box proteins, many of unknown function . We have disrupted a Dictyostelium discoideum gene, helC, which encodes helicase C, a member of the DEAD-box family of RNA helicases that shows strong homology to the product of the essential Saccharomyces cerevisiae gene dbp5 {2} and to related helicases in mouse and Schizosaccharomyces pombe . The HelC protein also shows weaker homology to the translation initiation factor elF-4a . Other DEAD-box-containing proteins, which are less closely related to HelC, have been implicated in developmental roles in Drosophila {3} and Xenopus laevis; one example is the Xenopus Vasa-like protein (XVLP) {4-6} . In Drosophila and Xenopus, Vasa and XVLP, respectively, are required for the establishment of tissue polarity during development . In yeast, DEAD-box helicases such as Prp8 {7} are components of the spliceosome and connect pre-mRNA splicing with the cell cycle . Disruption of the helC gene in D . discoideum led to developmental asynchrony, failure to differentiate and aberrant morphogenesis . We postulate that one reason for the existence of large families of homologous DEAD-box proteins in yeast, mammals and Dictyostelium could be that some DEAD-box proteins have developmentally specific roles regulating protein translation or mRNA stability.

J Cell Sci, 1998 Jun, 111 ( Pt 12), 1635 - 47
Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S . pombe; He X et al.; The spindle assembly checkpoint pathway is not essential for normal mitosis but ensures accurate nuclear division by blocking the metaphase to anaphase transition in response to a defective spindle . Here, we report the isolation of a new spindle checkpoint gene, mph1 (Mps1p-like pombe homolog), in the fission yeast Schizosaccharomyces pombe, that is required for checkpoint activation in response to spindle defects . mph1 functions upstream of mad2, a previously characterized component of the spindle checkpoint . Overexpression of mph1, like overexpression of mad2, mimics activation of the checkpoint and imposes a metaphase arrest . mph1 protein shares sequence similarity with Mps1p, a dual specificity kinase that functions in the spindle checkpoint of the budding yeast Saccharomyces cerevisiae . Complementation analysis demonstrates that mph1 and Mps1p are functionally related . They differ in that Mps1p, but not mph1, has an additional essential role in spindle pole body duplication . We propose that mph1 is the MPS1 equivalent in the spindle checkpoint pathway but not in the SPB duplication pathway . Overexpression of mad2 does not require mph1 to impose a metaphase arrest, which indicates a mechanism of spindle checkpoint activation other than mph1/Mps1p kinase-dependent phosphorylation . In the same screen which led to the isolation of mad2 and mph1, we also isolated dph1, a cDNA that encodes a protein 46% identical to an S . cerevisiae SPB duplication protein, Dsk2p . Our initial characterization indicates that S.p . dph1 and S.c . DSK2 are functionally similar . Together these results suggest that the budding and fission yeasts share common elements for SPB duplication, despite differences in SPB structure and the timing of SPB duplication relative to mitotic entry.

J Cell Sci, 1998 Jun, 111 ( Pt 12), 1603 - 12
The fission yeast microtubule cytoskeleton; Hagan IM; The Schizosaccharomyces pombe genome sequencing project is nearly complete, and this is likely to generate interest in fission yeast as a model system beyond its traditional strongholds in the study of the cell cycle and sexual differentiation . In many fields S . pombe will offer a useful complement to the more widely studied Saccharomyces cerevisiae, but in some areas the impact of S . pombe may well rival or exceed that of this budding yeast in terms of relevance to higher systems . Because of the considerable differences from the S . cerevisiae microtubule cytoskeleton, studying microtubules in S . pombe is likely to enhance the contribution of model systems to our understanding of the principles and practices of microtubule organisation in eukaryotes in general.

Genetics, 1998 May, 149(1), 117 - 30
The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa; Kothe GO et al.; Using an insertional mutagenesis approach, a series of Neurospora crassa mutants affected in the ability to control entry into the conidiation developmental program were isolated . One such mutant, GTH16-T4, was found to lack normal vegetative hyphae and to undergo constitutive conidiation . The affected gene has been named nrc-1, for nonrepressible conidiation gene #1 . The nrc-1 gene was cloned from the mutant genomic DNA by plasmid rescue, and was found to encode a protein closely related to the protein products of the Saccharomyces cerevisiae STE11 and Schizosaccharomyces pombe byr2 genes . Both of these genes encode MAPKK kinases that are necessary for sexual development in these organisms . We conclude the nrc-1 gene encodes a MAPKK kinase that functions to repress the onset of conidiation in N . crassa . A second mutant, GTH16-T17, was found to lack normal vegetative hyphae and to constitutively enter, but not complete, the conidiation program . The affected locus is referred to as nrc-2 (nonrepressible conidiation gene #2) . The nrc-2 gene was cloned and found to encode a serine-threonine protein kinase . The kinase is closely related to the predicted protein products of the S . pombe kad5, and the S . cerevisiae YNRO47w and KIN82 genes, three genes that have been identified in genome sequencing projects . The N . crassa nrc-2 gene is the first member of this group of kinases for which a phenotype has been defined . We conclude a functional nrc-2-encoded serine/threonine kinase is required to repress entry into the conidiation program.

Glycobiology, 1998 Jan, 8(1), 77 - 85
Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae; Eckert V et al.; The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library . The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified by complementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL . This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter . This construct allows to specifically suppress the endogenous enzyme activity . The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa . The deduced protein sequence shows a homology of over 90% when compared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species . This homology however decreases to 40-50% when compared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL . GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the recombinant plasmid and grown on glucose thus suppressing transcription of the endogenous gene . Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin . These results show that we have cloned the human GlcNAc-1-P transferase by heterologous complementation in S . cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.

Biochem Cell Biol, 1997, 75(6), 697 - 708
Markers of cell polarity during and after nitrogen starvation in Schizosaccharomyces pombe; Rupes I et al.; In Schizosaccharomyces pombe, nitrogen starvation induces transient acceleration of cell division and reduction in cell size with a final arrest in G1 . The division size control appears to be impaired by mutations in cdr1/nim1 and cdr2, genes that encode protein kinases mediating nutritional control over the mitotic cycle . cdr- cells arrest after fewer rounds of division and are larger than the wild type . Recent work suggests that long-term nitrogen starvation causes S . pombe wild-type cells to become spherical, which suggests loss of cell polarity . cdr mutants retain the elongated shape, indicating a potential difference in cell polarity control relative to the wild type . We examined several markers related to maintenance of cell polarity in S . pombe following nitrogen starvation including cell division scar pattern and actin and microtubule cytoskeleton . Wild-type cells as well as cdr mutants maintained a normal cell division scar pattern throughout nitrogen starvation but cells dividing under these conditions developed a wall malformation in the center of the septum . In cells arrested by nitrogen starvation, actin patches, normally associated with sites of cell wall deposition, were larger and distributed randomly along the cell surface . Cytoplasmic arrays of microtubules, which are thought to be involved in control of the polarity signal, were not visibly affected . The effects were similar in wild-type cells and in cdr- mutants . Upon refeeding, the new growth always reoccurred at the tip zones and there were only small deviations of its direction from the original axis . The results indicate that cell polarity is preserved both in wild-type cells, which arrest in G1 and appear spherical, and in cdr1/nim1 and cdr2 mutants, which arrest in G2 and appear polarized throughout the starvation period.

Curr Genet, 1998 Apr, 33(4), 255 - 61
Exogenous inositol and genes responsible for inositol transport are required for mating and sporulation in Shizosaccharomyces pombe; Niederberger C et al.; Fission yeast, Schizosaccharomyces pombe, is a natural inositol auxotroph . We show here that the amount of exogenous inositol added to the medium is critical for the control of its life cycle . Above growth-limiting concentrations inositol stimulates mating and sporulation in minimal medium . The effect of inositol is also observed on yeast-extract-medium plates . We selected a mutant, IM49, which mates and sporulates only poorly and show that it is defective in inositol transport . Its defect is in a gene (itr2) coding for a putative 12 membrane-spanning protein . The polypeptide contains the two sugar-transport motifs typical for hexose transporters and shows good homology to the two Saccharomyces cerevisiae inositol transporters . The itr2 gene is essential for cell growth and its mRNA level is repressed by glucose . Mutant IM49 is also complemented by a multicopy suppressor gene (itr1) which codes for a putative hexose transporter with unknown substrate specifity.

Curr Genet, 1998 Apr, 33(4), 248 - 54
S . pombe sck2+, a second homologue of S . cerevisiae SCH9 in fission yeast, encodes a putative protein kinase closely related to PKA in function; Fujita M et al.; The Schizosaccharomyces pombe sck2 gene, originally identified as SPAC22E12.14c in the genome-sequencing project, encodes a putative protein kinase highly similar to Saccharomyces cerevisiae Sch9p and S . pombe Sck1p, both of which can suppress loss of cAMP-dependent protein kinase (PKA) if over-produced . Over-expression of sck2 suppressed typical phenotypes of PKA-defective cells, including ectopic mating, slow growth and short cell morphology . Wild-type cells over-expressing sck2 behaved like the PKA-hyperactive mutant . Disruption of sck2 caused no obvious phenotype, but it intensified de-repression for sexual development when combined with the disruption of sck1 . The pka1 sck1 sck2 triple disruptant could grow but only very slowly . Whereas disruption of sck1 enhanced the inefficiency of Deltapka1 spores in germination, disruption of sck2 did not . These results suggest that the molecular function of Sck2p largely overlaps with that of Sck1p, but also that they differ somewhat either quantitatively or qualitatively.

FEBS Lett, 1998 Apr 17, 426(2), 283 - 9
Cloning and analysis of a novel human putative DNA methyltransferase; Van den Wyngaert I et al.; DNA methylation is intricately involved in a variety of cellular processes, such as differentiation, cell cycle progression, X-chromosome inactivation and genomic imprinting . However, little is known about how specific DNA methylation patterns are established and maintained . Previously one mammalian DNA methyltransferase has been described, but there has been considerable speculation about the presence of a second activity capable of methylation . Here we report the identification and characterization of a novel human putative DNA methyltransferase . Using a bioinformatics screen we have identified several expressed sequence tags which show high sequence similarity to the Schizosaccharomyces pombe gene pmt1+ . The cDNA for PuMet (for putative DNA methyltransferase) was cloned and the predicted amino acid sequence deduced . The gene is ubiquitously expressed, albeit at low levels . Like several other DNA methyltransferases, the bacterially overexpressed protein is not active in methylation assays.

Genomics, 1998 Apr 15, 49(2), 218 - 29
Rearrangement of the human CDC5L gene by a t(6;19)(p21;q13.1) in a patient with multicystic renal dysplasia; Groenen PM et al.; Genetic studies have implicated the short arm of chromosome 6 in congenital hydronephrosis . In previous studies, we described a fetus carrying a t(6;19)(p21;q13.1) as the sole cytogenetic anomaly and suffering from bilateral multicystic renal dysplasia caused by a bilateral complete pelviureteric junction obstruction, resulting in a massive hydronephrosis . Characterization of the chromosome 19 breakpoint region revealed that the transcription factor-encoding USF2 gene is affected . In this report, we show that the CDC5L gene on chromosome 6p is rearranged in the cells of the fetus . CDC5L encodes a protein that is related to the product of the Schizosaccharomyces pombe Cdc5 gene, which exerts its effects at the G2/M transition during cell cycle progression . We have established the genomic organization of the CDC5L gene and found that it consists of at least 16 exons spanning approximately 50 kb of chromosome segment 6p21 . Northern blot analysis indicated that the gene is ubiquitously expressed as a single mRNA of about 3.4 kb in both fetal and adult tissues . The translation product of the CDC5L gene has an electrophoretic mobility of about 100 kDa and is predicted to be a nuclear protein, since it contains a Myb-related DNA binding domain and potential nuclear localization signals in its aminoterminal region . Immunocytochemical analysis confirmed the nuclear localization of the CDC5L protein . CDC5L was also predicted to contain a hydrophilic, proline-rich region in its central part, which might function as a transcriptional activating domain . The chromosome 6 breakpoint was found in the intron between exons 9 and 10, indicating that, as a direct result of the 6;19 translocation, the Myb-related DNA binding domains and the nuclear localization signals are separated from the putative transactivating domain . Northern blot and RT-PCR experiments revealed that the other CDC5L allele is unaffected, and in Western blot experiments, expression of the 100-kDa protein was detected in fibroblasts of the fetus . Expression of a truncated or hybrid CDC5L transcript resulting from the CDC5L rearrangement could not be demonstrated.

Gene, 1998 Mar 27, 210(1), 143 - 50
Isolation of a novel heat shock protein 70-like gene, pss1+ of Schizosaccharomyces pombe homologous to hsp110/SSE subfamily; Chung KS et al.; A novel heat shock protein 70 (HSP70) gene, pss1+, of fission yeast, Schizosaccharomyces pombe (S . pombe), has been isolated as a multicopy suppressor of a synthetic lethal mutant of ras1+, which shows severe retardation of growth and aggregation phenotype when the ras1 gene function is absent . The pss1+ gene functionally complements the growth defect of the mutant . Sequence analysis revealed that pss1+ encodes an open reading frame (ORF) of 730amino acids that is homologous to the HSP70 family proteins . The Pss1 has high homology to the Saccharomyces cerevisiae (S . cerevisiae) heat shock protein Sse1p/Msi3p (43% identity) that belongs to the HSP110/SSE subfamily of HSP70 . The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of pss1+ gene . The transcript level of pss1+ was moderately abundant during steady-state growth at 25 degrees C and increased a few-fold upon shifting to 42 degrees C . Furthermore, transcription of pss1+ increased in nitrogen-starved conditions . Disruption of the pss1+ gene confers a temperature-sensitive growth phenotype and unexpectedly causes the increase in thermotolerance in S . pombe.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2598 - 605
Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA; Piard K et al.; We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene . We thus identified eight lethal mutants out of the 50 cDNAs tested . Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences . Two were full-length with a single point mutation at amino acid 68 or 69 . The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified . They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template . Cross-linking experiments showed that this was due to their inability to form trimers . Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.

J Cell Sci, 1998 Apr, 111 ( Pt 7), 867 - 76
F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe; Petersen J et al.; Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones . We describe the distribution of F-actin during sexual differentiation . Cortical F-actin dots have previously been shown to be restricted to one end of the rod shaped cell during the G1 phase of the cell cycle . Within half an hour of nitrogen starvation the distribution of cortical F-actin dots switched from being monopolar to bipolar . This was then reversed as the F-actin cytoskeleton repolarized so that cortical F-actin dots accumulated towards the projection tip at one end of the cell . Following cell fusion, F-actin dots were randomly scattered during the horsetail movement that precedes meiosis I and remained scattered until prometaphase or metaphase of meiosis II, when they concentrated around the nucleus . F-actin was seen on the lagging face of the nuclei which faced the partner nucleus during anaphase B of meiosis II . Early on in this anaphase F-actin was also seen on the opposite side of the nucleus, near the spindle pole body . F-actin accumulated within the spores in the mature ascus . Treatment with the actin depolymerising drug Latrunculin A showed that F-actin is required for cell fusion and spore formation . Latrunculin A treatment extended all stages from karyogamy to meiosis I . The S . pombe homologue of the actin binding protein profilin, Cdc3, was shown to be required for conjugation . Cdc3 co-localized with the formin related molecule Fus1 at the projection tip . The polarization of F-actin cortical dots to the projection tip was unaffected in the cdc3.124 mutant, but cdc3.124 mutant cells were unable to break down the cell walls between the two cells following agglutination.

J Cell Sci, 1998 Apr, 111 ( Pt 7), 853 - 65
Mutations in the bimC box of Cut7 indicate divergence of regulation within the bimC family of kinesin related proteins; Drummond DR et al.; Members of the bimC family of kinesin related proteins (KRPs) play vital roles in the formation and function of the mitotic spindle . Although they share little amino acid homology outside the highly conserved microtubule motor domain, several family members do contain a 'bimC box', a sequence motif around a p34(cdc2) consensus phosphorylation site in their carboxy-terminal 'tail' region . One family member, Eg5, requires phosphorylation at this site for association with the mitotic spindle . We show that mutations in the Schizosaccharomyces pombe cut7+ gene that change the bimC box p34(cdc2) consensus phosphorylation site at position 1,011 and a neighbouring MAP kinase consensus phosphorylation site at position 1,020 to non-phosphorylatable residues did not affect the ability of S . pombe cut7 genes to complement temperature sensitive cut7 mutants . Phosphorylation site mutants expressed as fusions to green fluorescent protein associated with the mitotic spindle with a localisation indistinguishable from similarly expressed wild-type Cut7 . Cells in which cut7.T1011A replaced the genomic copy of cut7+ were viable and formed normal spindles . Deletion of the entire carboxy-terminal tail region did not affect the ability of Cut7 to associate with the mitotic spindle but did inhibit normal spindle formation . Thus, unlike Eg5, neither the p34(cdc2) consensus phosphorylation site in the bimC box nor the entire tail region of Cut7 are required for association with the mitotic spindle.

Biochemistry, 1998 Apr 21, 37(16), 5542 - 8
Location of subunit-subunit contact sites on RNA polymerase II subunit 3 from the fission yeast Schizosaccharomyces pombe; Yasui K et al.; RNA polymerase II from the fission yeast Schizosaccharomyces pombe consists of 10 putative subunits . Subunit 3 (Rpb3) is a homologue of prokaryotic alpha subunit, which plays a key role in the assembly of core enzyme subunits . Previously we indicated that Rpb3 also plays an essential role in subunit assembly because it interacts with at least four subunits, two large subunits (Rpb1 and Rpb2) and two medium-sized subunits (Rpb3 and Rpb5) (1), and it constitutes a core subassembly consisting of Rpb2, Rpb3, and Rpb11 (2) . Using a synthetic mixture of equimolar amounts of individual subunits, which were all purified from cDNA-expressed Escherichia coli, we found here that Rpb3 also interacts with Rpb11, another alpha homologue . By making a set of Rpb3 deletion derivatives, we carried out mapping of the Rpb5- and Rpb11-contact sites on Rpb3 . By far-Western blot and GST pull-down assays, we found that the amino acid sequence between residues 105-263 of Rpb3 is involved in binding Rpb5, and the sequence between residues 105-297 is required for binding Rpb11 . Although the Rpb5- and Rpb11-contact sites on Rpb3 overlap each other, both subunits are able to associate with Rpb3 simultaneously . The binding of Rpb5 stabilizes the Rpb3-Rpb11 heterodimer.

J Biol Chem, 1998 Apr 10, 273(15), 8652 - 8
Processing of the presequence of the Schizosaccharomyces pombe Rieske iron-sulfur protein occurs in a single step and can be converted to two-step processing by mutation of a single proline to serine in the presequence; Nett JH et al.; The iron-sulfur proteins of the cytochrome bc1 complexes of Schizosaccharomyces pombe and Saccharomyces cerevisiae contain the three amino acid motif RX( downward arrow)(F/L/I)XX(T/S/G)XXXX (downward arrow) that is typical for proteins that are cleaved sequentially in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) . Despite the presence of this recognition sequence the S . pombe iron-sulfur protein is processed only once during import into mitochondria, whereas the S . cerevisiae protein is processed in two steps . Import of S . pombe iron-sulfur protein in which the putative MIP or MPP recognition sites are eliminated by site-directed mutagenesis and import of iron-sulfur protein into mitochondria from yeast mutants that lack MIP activity indicate that one step processing of the S . pombe iron-sulfur protein is independent of those sites and of MIP activity . Sequencing of the mature protein obtained after import in vitro and of the endogenous iron-sulfur protein isolated from mitochondrial membranes by preparative 2D-electrophoresis shows that MPP recognizes a second site in the presequence and processing occurs between residues 43 and 44 . If proline-20 of the S . pombe presequence is changed into a serine, a second cleavage step is induced . Conversely, if serine-24 of the S . cerevisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-sulfur protein to accumulate . Together these results indicate that a single amino acid change in the presequence is responsible for one-step processing in S . pombe versus two-step processing in S . cerevisiae.

Biochem Biophys Res Commun, 1998 Apr 7, 245(1), 246 - 53
Isolation and characterization of an invertase and its repressor genes from Schizosaccharomyces pombe; Tanaka N et al.; PCR was used to isolate an invertase homolog gene from the fission yeast Schizosaccharomyces pombe . The cloned inv1(+) gene encodes a protein of 581 amino acids with 16 potential asparagine-linked glycosylation sites, and has 39% and 38% identity to the Schwanniomyces occidentalis and Saccharomyces cerevisiae SUC2 invertases . When the inv1(+) gene was disrupted, S . pombe strains lacked detectable invertase activity . This result showed that the inv1(+) gene encodes only one active invertase in S . pombe cells . The transcription of inv1(+) is repressed in the presence of glucose . The transcription of inv1(+) was not affected in cyr1Delta strain which lacks adenylate cyclase activity, unlike transcription of S . pombe fbp1(+) gene . We have identified an S . pombe gene (scr1(+)) that encodes a homolog of the Aspergillus nidulans CREA which is required for glucose repression of the glyconeogenic pathway . Although the deletion of scr1(+) did not influence the transcription of fbp1(+) gene, glucose repression of the inv1(+) gene was severely affected . These results showed that glucose repression of inv1(+) gene is dependent on scr1(+) gene, and S . pombe cAMP signalling pathway may not be essential for glucose repression of inv1(+) gene .

Biochem Biophys Res Commun, 1998 Apr 7, 245(1), 166 - 71
Secretion of heterologous proteins from Schizosaccharomyces pombe using the homologous leader sequence of pho1+ acid phosphatase; Braspenning J et al.; In this study we report the use of the S . pombe leader sequence of pho1+ acid phosphatase (Elliott et al., J . Biol . Chem . 216, 2916-2941, 1986) for the secretion of heterologous proteins into the medium . The green fluorescent protein (GFP) and the Human Papillomavirus (HPV) type 16 E7 protein are normally not secreted; fusion of the S . pombe pho1 leader peptide (SPL) to GFP and HPV 16 E7 resulted in an efficient secretion of these proteins although the latter contains a nuclear targeting sequence . These data suggest that SPL fused constructs could be applied for the production of other recombinant proteins using the S . pombe expression system . Furthermore, since GFP retains its intrinsic fluorescence during the secretion, this system may be useful to study the secretory pathway of fission yeast in vivo .

Oncogene, 1998 Apr 2, 16(13), 1759 - 65
Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast; Waddell S et al.; The majority of human anogenital carcinomas show evidence of papillomavirus infection . To facilitate viral replication, viruses disable key cellular responses which would otherwise precipitate cell suicide . An obligate factor in one such response is the p53 tumour suppressor protein . p53 gene mutation is an infrequent event in anogenital cancer, apparently due to the action of HPV E6 protein, which inhibits wild-type p53 function by stimulating the degradation of p53 protein . p53 is required for the apoptotic response that is triggered in untransformed cells following inappropriate cell-cycling . E6 directed inhibition of p53 function thus facilitates the survival of transformed cells . We have developed a genetically tractable model that reports E6 protein-mediated human p53 inactivation in the fission yeast Schizosaccharomyces pombe . Functional dissection of the requirements for E6 directed inhibition in this system reveal an absolute requirement for the presence of both E6 protein and the human E3 ubiquitin ligase, E6-AP . Using a defined set of E6 mutants we show that degradation of p53 protein rather than E6/p53 association is likely required for E6-mediated inhibition . This S . pombe based system represents a candidate screen for novel antiviral agents that act by disrupting the E6/E6-AP/p53 interaction.

Appl Microbiol Biotechnol, 1998 Mar, 49(3), 301 - 8
Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products; Okada H et al.; A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast . Schizosaccharomyces pombe . The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase . The transformed S . pombe strain produced over 115 micrograms cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 micrograms/ml gentamicin . The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) . Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to alpha-mannosidase . The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T . reesei in their optimum pH and temperature, pH and temperature stabilities, and Vmax values toward phosphoric-acid-swollen cellulose as substrate, except that their Km values were about four-fold higher than that of the native enzyme.

Microbiology, 1998 Apr, 144 ( Pt 4), 1085 - 93
Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants; Zhou GL et al.; The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization . This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes . The L . edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein . The L . edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively . The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity) . Southern blotting and Northern blotting results suggest that L . edodes cap is a single-copy gene and uniformly expressed . Expression of the L . edodes CAP in both Schiz . pombe and Sacch . cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP . By using a yeast two-hybrid assay, an interaction was demonstrated between the L . edodes CAP and Schiz . pombe actin . This result and the functional complementation test indicate that CAP from L . edodes has a conserved C-terminal domain function.

J Cell Sci, 1998 Mar, 111 ( Pt 6), 843 - 51
The Cdk inhibitors p25rum1 and p40SIC1 are functional homologues that play similar roles in the regulation of the cell cycle in fission and budding yeast; Sanchez-Diaz A et al.; p25rum1 and p40SIC1 are specific inhibitors of p34(cdc2/CDC28) kinase complexes with B-type cyclins that play a central role in the regulation of the G1 phase of the cell cycle . We show here that low levels of expression of SIC1 in Schizosaccharomyces pombe rescues all the phenotypes of cells lacking the rum1+ gene . In addition, high level expression of SIC1 in S . pombe induces extra rounds of DNA replication without mitosis, a phenotype very similar to the overexpression of rum1+ . Transient expression of rum1+ in S . cerevisiae restores the G1 arrest phenotype of cdc4 sic1Delta double mutants . Overproduction of rum1+ in Saccharomyces cerevisiae causes a cell cycle block in G1 with a phenotype similar to inactivation of all the Clb cyclins . Finally, we have mapped the cyclin interacting domain and Cdk inhibitory domain to a region of about 80 amino acids in p25rum1 that has significant homology to the C-terminal domain of p40SIC1 . All these observations suggest that fission yeast p25rum1 and budding yeast p40SIC1 define a family of Cdk inhibitors that specifically down regulate cyclin B/Cdk1 during the G1 phase of the cell cycle.

J Cell Sci, 1998 Mar, 111 ( Pt 6), 701 - 12
Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells; Ding DQ et al.; Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe . Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions . To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively . Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body . During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell . When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules . Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement . The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute . We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

Nature, 1998 Apr 23, 392(6678), 825 - 8
Defective meiosis in telomere-silencing mutants of Schizosaccharomyces pombe; Nimmo ER et al.; During meiotic prophase, chromosomes frequently adopt a bouquet-like arrangement, with their telomeres clustered close to the nuclear periphery . A dramatic example of this occurs in the fission yeast, Schizosaccharomyces pombe, where all telomeres aggregate adjacent to the spindle pole body (SPB) . Nuclei then undergo rapid traverses of the cell, known as 'horsetail' movement, which is led by the SPB dragging telomeres and chromosomes behind . This process may initiate or facilitate chromosome pairing before recombination and meiosis . With the aim of identifying components involved in telomere structure and function, we report here the isolation of S . pombe mutants defective in the ability to impose transcriptional silencing on genes placed near telomeres . Two of these mutants, lot2-s17 and lot3-uv3, also display a dramatic lengthening of telomeric repeats . lot3-uv3 carries a mutation in Taz1, a telomere-binding protein containing a Myb-like motif similar to two human telomere-binding proteins . Meiosis is aberrant in these mutant yeast strains, and our analysis demonstrates a decreased association of telomeres with the SPB in meiotic prophase . This results in defective 'horsetail' movement, a significant reduction in recombination, low spore viability and chromosome missegregation through meiosis.

DNA Cell Biol, 1998 Apr, 17(4), 349 - 58
Identical cis-acting elements and related trans-acting factors control activity of nonviral promoter in Schizosaccharomyces pombe and mammalian cells; Brys R et al.; We have analyzed the transcriptional activity of the human plasminogen activator inhibitor-1 promoter in the fission yeast Schizosaccharomyces pombe . This promoter is active in S . pombe, and the initiation site of transcription corresponds to the site identified previously in mammalian cells . Mutations in the AP-1-binding site (PAI-1 A box) or the HLTF-binding site (the B box), which reduced the basal and phorbol ester-induced levels of PAI-1 expression in human cells, also decreased the transcriptional activity in S . pombe . Gel retardation assays showed that an S . pombe protein binds specifically to this B box element and displays the same B box sequence requirement as HLTF . Furthermore, this yeast protein binds specifically to other HLTF-binding sites in the human immunodeficiency virus-1 long terminal repeat (LTR) and the simian virus 40 (SV40) enhancer . The B box (but not a mutated B box) strongly stimulated transcription when combined with adh downstream promoter elements, indicating that the S . pombe B box-binding protein, like HLTF, is a transcriptional activator . We conclude that the transcriptional activity of the nonviral PAI-1 promoter is controlled by the same promoter elements in S . pombe as in mammalian cells . In addition, mammalian trans-acting factors that bind to these promoter elements were shown to have counterparts with conserved DNA-binding activity in S . pombe . These results further illustrate the conservation of the mechanism of transcription between mammalian cells and fission yeast.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 145 - 50
Growth phase-dependent active transport of pyridoxine in a fission yeast, Schizosaccharomyces pombe; Yagi T et al.; Schizosaccharomyces pombe showed maximum pyridoxine uptake activity around 10 h after starting cultivation . High concentrations of thiamine and pyridoxine in the medium did not affect the activity or the time but changed intracellular levels of vitamin B6 compounds . Pyridoxine was taken up by a saturable mechanism with two kinds of affinity (K(m) 22.4 microM and 118 microM) . The uptake depended on the energy produced anaerobically with an optimum pH of 4.5 . The uptake was completely inhibited by amiloride, sodium azide or 2,4-dinitrophenol . The uptake system of the fission yeast was different in various respects from that of a budding yeast.

Genetics, 1998 Apr, 148(4), 1731 - 42
Sum1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast; Humphrey T et al.; The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication . In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal . To isolate genes that function in the checkpoint pathway, we screened an S . pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w . Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis) . sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae . S . pombe sum1+ is an essential gene, required for normal cell growth and division . In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress . Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells . These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast.

Yeast, 1998 Mar 30, 14(5), 485 - 92
Characterization of the Prk1 protein kinase from Schizosaccharomyces pombe; Watson P et al.; We report the isolation and characterization of a protein kinase from the fission yeast Schizosaccharomyces pombe . The proposed Prk1 protein contains 352 amino acids and has significant homology to the Ume5p kinase (also known as Srb10p, Ssn3p and Are1p) of the budding yeast Saccharomyces cerevisiae, a cyclin-dependent kinase involved in regulating the transcription of a diverse set of genes . Disruption of the prk1 gene increases flocculation but does not appear to have any other significant effect on cell behaviour . This defect can be overcome by expressing the UME5 gene, indicating that Prk1 is the fission yeast homologue of Ume5p.

Yeast, 1998 Mar 30, 14(5), 479 - 84
Nucleotide sequence of the Schizosaccharomyces pombe lys1+ gene and similarities of the lys1+ protein to peptide antibiotic synthetases; Bhattacherjee V et al.; The 4.2 kbp lys1+ gene of Schizosaccharomyces pombe encoding the large subunit of alpha-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H . The S . pombe lys1+ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155.8 kDa . The encoded protein converts alpha-aminoadipic acid to alpha-aminoadipate-delta-semialdehyde by an ATP-mediated adenylation . Analysis of the sequence showed that the putative protein encoded by lys1+ shares strong homology with the peptide antibiotic synthetases which also use in adenylation step.

J Bioenerg Biomembr, 1997 Dec, 29(6), 603 - 9
Estimation of membrane potential deltapsi in reconstituted plasma membrane vesicles using a numerical model of oxonol VI distribution; Portele A et al.; A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference deltapsi across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed . deltapsi was generated by the H+-ATPase reconstituted in the PMV . The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K+ + valinomycin) failed . The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding deltapsi in ATP-energized PMV of 80 mV . The described model treatment to estimate deltapsi may be applicable for other reconstituted membrane systems.

Plant Cell Physiol, 1998 Feb, 39(2), 131 - 8
Cloning and characterization of high-CO2-specific cDNAs from a marine microalga, Chlorococcum littorale, and effect of CO2 concentration and iron deficiency on the gene expression; Sasaki T et al.; Two cDNA clones exclusively induced under an extremely high-CO2 concentration (20%) were isolated from Chlorococcum littorale by differential screening and named HCR (high-CO2 response) 1 and 2, respectively . The amino acid sequence of the protein encoded by HCR2 exhibited homology to the gp91-phox protein, a critical component of a human phagocyte oxidoreductase, and to the yeast ferric reductases, Saccharomyces cerevisiae FRE1 and FRE2 and Schizosaccharomyces pombe Frp1 . The induction of both HCR mRNAs required extremely high-CO2 conditions and iron deficiency, being suppressed under air conditions and by iron sufficiency, suggesting that the expression of these two HCR genes required extremely high-CO2 conditions and iron deficiency in combination . The HCR2 protein was detected in the membrane fractions of cells grown under conditions which would favor the induction of HCR2-mRNA and the protein level was lowered when the cells were transferred from iron deficient to 10 microM FeSO4 conditions (with 20% CO2).

Bioorg Khim, 1998 Jan, 24(1), 42 - 7
{Exon-intron structure of the fet5+ gene of Schizosaccharomyces pombe and physical mapping of genome encompassing regions}; Shpakovskii GV et al.; Plasmid pYUK3 bearing the fet5+ gene of Schizosaccharomyces pombe was isolated from a genomic library of the fission yeast, and a detailed physical map of the whole genomic insert (ca . 9.6 Kbp) was constructed . The primary structure of the fet5+ gene and its flanking regions is established . The gene contains a single 45-bp intron in its distal part . A typical TATA-box (TATAAG) was found in the 5'-noncoding region ca . 50 bp upstream of the putative start of transcription, and the 3'-noncoding region contains AT-rich palindromes, which are probably involved in termination of the fet5+ transcription . A previously unidentified gene of Sz . pombe encoding a protein with some similarity to one of the transcriptional activators from the TBP (TATA-binding protein) group of SPT factors of transcription was found in the vicinity of the fet5+ gene . Taking into account that cDNA of the fet5(+)-gene was isolated as a suppressor of the genetic-defect of nuclear RNA polymerases I-III (Bioorg . Khim., 1997, vol . 23, No 3, pp . 234-237), this vicinity may be the first evidence of possible clustering, in the genome of the fission yeast, of genes participating in transcription regulation.

J Chromatogr B Biomed Sci Appl, 1998 Feb 27, 706(1), 113 - 21
Purification of a recombinant protein expressed in yeast: optimization of analytical and preparative chromatography; Raymond F et al.; The industrial production of recombinant proteins requires control of both fermentation and purification steps . For the serodiagnosis of toxoplasmosis, the main antigen is a membrane protein of 30 kDa (P30) . The P30 gene was cloned and expressed in Schizosaccharomyces pombe at 0.7 microg/ml in culture medium . Batch fermentation was optimized by the specific choice of peptones, which enabled optimum growth and protein expression without reducing the efficacy of the purification step . Analytical purification was then carried out using cation-exchange chromatography . For larger volumes, scaling up was performed on expanded mode by using a Streamline system (Pharmacia) . This purification step allowed us to obtain a 67.5% recovery with a purification factor greater than 27-fold . Expanded bed adsorption technology is a convenient and effective technique for protein capture directly from feedstock, and the eluted fraction is ready for a second affinity chromatography step . This second step is performed with a yield of 40% and provides a final purification factor of 2000-fold.

J Biol Chem, 1998 Mar 6, 273(10), 5869 - 77
Temperature-sensitive mutations of the CKA1 gene reveal a role for casein kinase II in maintenance of cell polarity in Saccharomyces cerevisiae; Rethinaswamy A et al.; Casein kinase II (CKII) of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', that are encoded by the CKA1 and -2 genes, respectively . We have constructed conditional alleles of the CKA1 gene . In contrast to cka1 cka2(ts) strains, which exhibit a defect in both G1 and G2/M cell cycle progression, cka1(ts) cka2 strains continue to divide for three cell cycles after a shift to restrictive temperature and then arrest as a mixture of budded and unbudded cells with a spherical morphology . Arrested cells exhibit continued growth, a nonpolarized actin cytoskeleton, delocalized chitin deposition, and a significant fraction of multinucleate cell bodies, confirming the presence of a cell polarity defect in cka1(ts) strains . The presence of budded as well as unbudded cells in the arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both processes is also possible . The terminal phenotype of cka1(ts) strains bears a strong resemblance to that of orb5 strains of Schizosaccharomyces pombe, which carry a temperature-sensitive CKII catalytic subunit mutation, but the underlying mechanism appears to be different in the two cases . These results establish a requirement for CKII in cell polarity in S . cerevisiae and provide the first evidence for functional specialization of CKA1 and -2.

J Biol Chem, 1998 Mar 6, 273(10), 5765 - 70
Tyrosine phosphorylation regulates the SH3-mediated binding of the Wiskott-Aldrich syndrome protein to PSTPIP, a cytoskeletal-associated protein; Wu Y et al.; Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system . Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton . Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p . This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP . Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain . Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation . One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo . In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro . These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton.

Curr Biol, 1998 Jan 29, 8(3), 135 - 44
Regulated vacuole fusion and fission in Schizosaccharomyces pombe: an osmotic response dependent on MAP kinases; Bone N et al.; BACKGROUND: The budding yeast Saccharomyces cerevisiae uses two mitogenactivated protein (MAP) kinase cascades, the Hog1p and the Mpk1p pathways, to signal responses to hypertonic and hypotonic stress, respectively . Mammalian cells and the fission yeast Schizosaccharomyces pombe have functional homologues of Hog1p - p38/RK/CSBP and Sty1 - which, unlike Hog1p, also mediate other responses . We have investigated the involvement of S . pombe MAP kinase pathways in signalling a newly described response to osmotic stress - that of vacuole fusion and fission . RESULTS: When S . pombe is placed into water, its vacuoles rapidly fuse into larger structures enclosing a greater proportion of the cell's volume . Under some conditions, its vacuoles can slowly fragment in response to salt . Fission requires the Sty1 pathway and also Pmk1, the homologue of S . cerevisiae Mpk1p . Fusion requires Pmk1, Ypt7 - the homologue of a protein involved in S . cerevisiae vacuole fusion - and part of the Sty1 pathway, although Sty1 phosphorylation is unaffected by hypotonic conditions . CONCLUSIONS: Vacuole fusion and fission appear to be homeostatic mechanisms that restore the concentration of the cytosol . Vacuole fusion, like stimulated secretion in higher eukaryotes, is a rapid and specific process of membrane fusion in response to an external stimulus . The Sty1 pathway, in addition to its role in responding to hypertonic stress, is required at a basal level for the expression of factors required to respond to hypotonic stress - a mechanism that may allow the cell to use a common pathway for different responses.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 390 - 2
Multidrug resistance phenotype conferred by overexpressing bfr2+/pad1+/sks1+ or pap1+ genes and mediated by bfr1+ gene product, a structural and functional homologue of P-glycoprotein in Schizosaccharomyces pombe; Arioka M et al.; We investigated the mechanism of multidrug resistance conferred by overexpression of bfr2+/pad1+/sks1+ or pap1+ genes of Schizosaccharomyces pombe . Overexpression of bfr2+ did not confer multidrug resistance on a pap1-disrupted strain . In a mutant with bfr1+ (a putative membrane transporter which belongs to the ATP-binding cassette superfamily) disrupted, overexpression of either bfr2+ or pap1+ did not confer multidrug resistance . These findings suggest that bfr1+ acts as the most downstream effector of the multidrug resistance conferred by bfr2+ and pap1+ genes.

Microbiol Mol Biol Rev, 1998 Mar, 62(1), 55 - 70
Molecular genetics of mating recognition in basidiomycete fungi; Casselton LA et al.; The recognition of compatible mating partners in the basidiomycete fungi requires the coordinated activities of two gene complexes defined as the mating-type genes . One complex encodes members of the homeobox family of transcription factors, which heterodimerize on mating to generate an active transcription regulator . The other complex encodes peptide pheromones and 7-transmembrane receptors that permit intercellular signalling . Remarkably, a single species may have many thousands of cross-compatible mating types because the mating-type genes are multiallelic . Different alleles of both sets of genes are necessary for mating compatibility, and they trigger the initial stages of sexual development--the formation of a specialized filamentous mycelium termed the dikaryon, in which the haploid nuclei remain closely associated in each cell but do not fuse . Three species have been taken as models to describe the molecular structure and organization of the mating-type loci and the genes sequestered within them: the pathogenic smut fungus Ustilago maydis and the mushrooms Coprinus cinereus and Schizophyllum commune . Topics addressed in this review are the roles of the mating-type gene products in regulating sexual development, the molecular basis for multiple mating types, and the molecular interactions that permit different allelic products of the mating type genes to be discriminated . Attention is drawn to the remarkable conservation in the mechanisms that regulate sexual development in basidiomycetes and unicellular ascomycete yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, a theme which is developed in the general conclusion to include the filamentous ascomycetes Neurospora crassa and Podospora anserina.

Mol Cell Biol, 1998 Apr, 18(4), 2118 - 29
The Schizosaccharomyces pombe mei4+ gene encodes a meiosis-specific transcription factor containing a forkhead DNA-binding domain; Horie S et al.; The mei4+ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation . The mei4 disruptant failed to complete meiosis-I but could proliferate normally . mei4+ was transcribed only in meiosis-proficient diploid cells after premeiotic DNA replication . The mei4+ open reading frame encodes a 57-kDa serine-rich protein comprised of 517 amino acids with a forkhead/HNF3 DNA-binding domain in the amino-terminal region . Transcription of spo6+, a gene required for sporulation, was dependent on the mei4+ function . Two copies of the GTAAAYA consensus sequence, proposed as the binding site for human forkhead proteins, were found in the promoter region of spo6+ . A gel mobility shift assay demonstrated the sequence-dependent binding of the GST-Mei4 forkhead domain fusion protein to DNA fragments with one of the consensus elements . Deletion of this consensus element from the spo6 promoter abolished the transcription of spo6+ and resulted in a sporulation deficiency . One-hybrid assay of Mei4 which was fused to the Gal4 DNA-binding domain localized the transcriptional activation domain in the C-terminal 140 amino acids of Mei4 . These results indicate that Mei4 functions as a meiosis-specific transcription factor of S . pombe.

Am J Respir Cell Mol Biol, 1998 Mar, 18(3), 297 - 306
Pneumocystis carinii contains a functional cell-division-cycle Cdc2 homologue; Thomas CF et al.; Pneumocystis carinii causes life-threatening pneumonia in immunocompromised patients . The inability to culture P . carinii has hampered basic investigations of the organism's life cycle, limiting the development of new therapies directed against it . Recent investigations indicate that P . carinii is a fungus phylogenetically related to other ascomycetes such as Schizosaccharomyces pombe . The cell cycles of S . pombe and homologous fungi are carefully regulated by cell-division-cycle molecules (cdc), particularly cell-division-cycle 2 (Cdc2), a serine-threonine kinase with essential activity at the G1 restriction point and for entry into mitosis . Antibodies to the proline-serine-threonine-alanine-isoleucine-arginine (PSTAIR) amino-acid sequence conserved in Cdc2 proteins specifically precipitated, from P . carinii extracts, a molecule with kinase activity consistent with a Cdc2-like protein . Cdc2 molecules exhibit differential activity throughout the life cycle of the organisms in which they occur . In accord with this, the P . carinii Cdc2 showed greater specific activity in P . carinii trophic forms (trophozoites) than in spore-case forms (cysts) . In addition, complete genomic and complementary DNA (cDNA) sequences of P . carinii Cdc2 were cloned and found to be most closely homologus to the corresponding sequences of other pathogenic fungi . The function of P . carinii cdc2 cDNA was further documented through its ability to complement the DNA of mutant strains of S . pombe with temperature-sensitive deficiencies in Cdc2 activity . The P . carinii cdc2 cDNA restored normal Cdc2 function in these mutant strains of S . pombe, and promoted fungal proliferation . These studies represent the first molecular analysis of the cell-cycle-regulatory machinery in P . carinii . Further understanding of P . carinii's life cycle promises novel insights for preventing and treating the intractable infection it causes in immunocompromised patients.

Mol Biol Cell, 1998 Mar, 9(3), 611 - 21
Regulation of telomere length by checkpoint genes in Schizosaccharomyces pombe; Dahlen M et al.; We have studied telomere length in Schizosaccharomyces pombe strains carrying mutations affecting cell cycle checkpoints, DNA repair, and regulation of the Cdc2 protein kinase . Telomere shortening was found in rad1, rad3, rad17, and rad26 mutants . Telomere lengths in previously characterized rad1 mutants paralleled the replication checkpoint proficiency of those mutants . In contrast, rad9, chk1, hus1, and cds1 mutants had intact telomeres . No difference in telomere length was seen in mutants affected in the regulation of Cdc2, whereas some of the DNA repair mutants examined had slightly longer telomeres than did the wild type . Overexpression of the rad1(+) gene caused telomeres to elongate slightly . The kinetics of telomere shortening was monitored by following telomere length after disruption of the rad1(+) gene; the rate was approximately 1 nucleotide per generation . Wild-type telomere length could be restored by reintroduction of the wild-type rad1(+) gene . Expression of the Saccharomyces cerevisiae RCK1 protein kinase gene, which suppresses the radiation and hydroxyurea sensitivity of Sz . pombe checkpoint mutants, was able to attenuate telomere shortening in rad1 mutant cells and to increase telomere length in a wild-type background . The functional effects of telomere shortening in rad1 mutants were assayed by measuring loss of a linear and a circular minichromosome . A minor increase in loss rate was seen with the linear minichromosome, and an even smaller difference compared with wild-type was detected with the circular plasmid.

Curr Genet, 1998 Jan, 33(1), 38 - 45
Isolation and characterization of the aureobasidin A-resistant gene, aur1R, on Schizosaccharomyces pombe: roles of Aur1p+ in cell morphogenesis; Hashida-Okado T et al.; To study the mechanism of action of the antibiotic aureobasidin A (AbA) on yeasts, we isolated a dominant mutant of Schizosaccharomyces pombe which gave high resistance to AbA . From a genomic library of the mutant, an aur1R mutant gene conferring AbA resistance was isolated . One amino-acid mutation, a substitution of glycine with cysteine at residue 240, was responsible for the acquisition of AbA resistance . The wild-type aur1+ gene was essential for viability, and its over-expression enhanced significant resistance to AbA . The predicted protein of S . pombe aur1R was highly homologous in primary structure and hydropathy profile with that of Saccharomyces cerevisiae AUR1R isolated as an AbA-resistance gene . To analyze a role in cell growth of S . pombe aur1+, temperature-sensitive mutants (aur1ts) were obtained by random mutagenesis procedures using a modified PCR . The aur1ts mutation caused a defect in cell elongation at the non-permissive temperature and finally led to cell death . These results suggest that Aur1p was a target of the antibiotic AbA and was required in the cell elongation of cell-end tips and in the viability of S . pombe.

Curr Genet, 1998 Jan, 33(1), 29 - 37
The Ste16 WD-repeat protein regulates cell-cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe; Maekawa H et al.; The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation) . We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation . Genetic analysis of these mutants defined a single locus designated as ste16 . The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half . The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift . When transferred to fresh growth medium, the G2-arrested ste16Delta haploids resumed the mitotic cycle from the S-phase, resulting in diploidization . This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase . As the Rum1 protein level was remarkably elevated in the ste16Delta, the Ste16 protein negatively controls the Rum1 level . The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy.

Biochim Biophys Acta, 1998 Mar 4, 1396(1), 15 - 20
Schizosaccharomyces pombe apn1 encodes a homologue of the Escherichia coli endonuclease IV family of DNA repair proteins; Ramotar D et al.; The Apn1 protein of the budding yeast Saccharomyces cerevisiae is a DNA repair enzyme that hydrolyzes apurinic/apyrimidinic (AP) sites and removes 3'-blocking groups present at single strand breaks of damaged DNA . Yeast cells lacking Apn1 are hypersensitive to DNA damaging agents that produce AP sites and DNA strand breaks with blocked 3'-termini . In this study, we showed that the fission yeast Schizosaccharomyces pombe bears a homologue, Spapn1, that is 45% identical to S . cerevisiae Apn1 . However, the Spapn1 gene is apparently not expressed . Active expression of S . cerevisiae Apn1 in S . pombe conferred no additional resistance to DNA damaging agents . These data suggest that the pathway by which S . pombe repairs AP sites is independent of a functional Apn1-like AP endonuclease.

Nature, 1998 Mar 19, 392(6673), 303 - 6
Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast; Mizunuma M et al.; Signalling via calcium is probably involved in regulating eukaryotic cell proliferation, but details of its mechanism of action are unknown . In Schizosaccharomyces pombe, the onset of mitosis is determined by activation of a complex of the p34cdc2 protein kinase and a cyclin protein that is specific to the G2 phase of the cell cycle . This activation requires dephosphorylation of p34cdc2 . Weel, a tyrosine kinase that inhibits p34cdc2 by phosphorylating it, is needed to determine the length of G2 phase . Here we show that calcium-activated pathways in Saccharomyces cerevisiae control the onset of mitosis by regulating Swel, a Weel homologue . Zds1 (also known as Oss1 and Hst1) is important in repressing the transcription of SWE1 in G2 phase . In the presence of high calcium levels, cells lacking Zds1 are delayed in entering mitosis . Calcineurin and Mpk1 regulate Swel activation at the transcriptional and posttranslational levels, respectively, and both are required for the calcium-induced delay in G2 phase . These cellular pathways also induce a G2-phase delay in response to hypotonic shock.

Cell Motil Cytoskeleton, 1998, 39(3), 195 - 200
Yeast myosin II: a new subclass of unconventional conventional myosins?
May KM, Win TZ, Hyams JS.
Myosin II is the founder member of a large and structurally diverse clan of actin-based motor proteins . The native myosin II molecule is a hexamer consisting of two heavy chains, two essential light chains (ELC), and two regulatory light chains (RLC) . For convenience, the myosin IIs are often subdivided into four subclasses: vertebrate skeletal and cardiac muscle myosin II form one subclass, vertebrate smooth muscle and nonmuscle myosin II a second, invertebrate muscle a third, and protozoan myosin II a fourth {Sellers and Goodson, 1995} . Different mechanisms of regulation may exist between myosins within a single subclass yet all myosin IIs share a common three-domain structure; the N-terminus of the heavy chain forms two globular heads that contain the ATP- and actin-binding sites and the alpha-helical neck region that is stabilised by the binding of the two classes of light chains, whilst the C-terminus forms an extended coiled-coil tail that can consist of anywhere between 700 and 1,200 amino acids . In nonmuscle cells, myosin II has at least two well-defined functions, cell locomotion and cytokinesis . Yeast cells do not locomote, and their mechanism of cytokinesis involves the deposition of a cross-wall or septum . However, in the fission yeast, Schizosaccharomyces pombe, deposition of the septum is anticipated by the appearance of a contractile actomyosin ring {Marks and Hyams, 1985; May et al., 1997; Kitayama et al., 1997} and actin is also present at the bud neck during cytokinesis in the budding yeast, Saccharomyces cerevisiae {Kilmartin and Adams, 1984} . Here we report a phylogenetic analysis of the N-terminal head domains of the myosin IIs from both yeasts, a structural analysis of the tail domains of these proteins and we speculate as to the nature of the light chains that regulate their function . On the basis of these findings, we propose that the yeast myosin IIs constitute a divergent fifth class of "unconventional" conventional myosins.

Mol Gen Genet, 1998 Feb, 257(3), 319 - 29
Isolation and characterization of hrp1+, a new member of the SNF2/SWI2 gene family from the fission yeast Schizosaccharomyces pombe; Jin YH et al.; The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair . Several proteins with unknown functions are also included in this family . The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members . This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family . The hrp1+ (helicase-related gene from S . pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair . The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa . Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain . This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S . pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression . Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually . DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+ . Deletion of the hrp1+ gene resulted in accelerated cell growth . On the other hand, overexpression of Hrp1 caused a reduction in growth rate . These results indicate that hrp1+ may act as a negative regulator of cellular growth.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 546 - 50
Identification of Schizosaccharomyces pombe prenol as dolichol-16,17; Quellhorst GJ Jr et al.; The identity of the prenol involved in N-linked glycosylation in the fission yeast Schizosaccharomyces pombe was unknown . In order to determine the identity of the prenol, S . pombe cells were incubated with a metabolic precursor of prenol, tritiated mevalonolactone . The cells incorporated only a modest amount of label, about 1000 dpm per million cells, into base-stable lipid and only 1% of that radioactivity was incorporated into prenol . We found by normal phase silica HPLC and more directly by the lack of reactivity with MnO2 that the labeled lipid was predominantly dolichol, not polyprenol . Reverse phase HPLC demonstrated that in S . pombe dolichol ranged between 14 and 18 isoprene units with dolichol-16,17 being the most abundant prenol . This dolichol is of an intermediate length, between the dolichol of S . cerevisiae and that of mammalian cells.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 468 - 74
The Byr2 kinase translocates to the plasma membrane in a Ras1-dependent manner; Bauman P et al.; The activation of mitogen-activated protein kinase cascades by the Ras GTPase is an evolutionarily conserved signal transduction mechanism . To better understand the interaction between Ras and its target kinase, we study the yeast Schizosaccharomyces pombe where the Ras1 GTPase activates the Byr2 kinase . Cell fractionation and immunofluorescence showed that Ras1 was localized to the plasma membrane and that Byr2 was in the cytoplasm . When Ras1 was overexpressed, Byr2 was translocated to the plasma membrane . Byr2 translocation was dependent on binding to Ras1 since Ras1-V12, an activated mutant of Ras1, caused more Byr2 translocation than Ras1, since Ras1-D38E, an effector domain mutant, did not cause Byr2 translocation, and since the Ras1-binding domain of Byr2 was necessary and sufficient to cause Byr2 translocation . The Byr2 protein was usually not uniform around the plasma membrane, but was frequently enriched at the cell ends and at the region of septal deposition . This uneven membrane localization depended upon regions of the Byr2 regulatory domain, in addition to those required for Ras1 binding, suggesting that these Byr2 domains participate in protein-protein interactions.

Mol Cells, 1997 Dec 31, 7(6), 800 - 6
Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe; Chung KS et al.; Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms . Various cellular factors are known to interact with Ras proteins . In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1 . We first constructed S . pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine . This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine . Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-) . One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei . The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent . These mutants, however, grow normally when Ras1 is expressed (ras1+) . These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S . pombe . Screening of a genomic library of S . pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S . pombe.

DNA Res, 1997 Dec 31, 4(6), 393 - 6
Identification and characterization of a novel trans-membrane protein gene, pdh1, from Schizosaccharomyces pombe; Iha H et al.; We have cloned a new gene, pdh1, from genomic DNA of fission yeast Schizosaccharomyces pombe . pdh1 is actively transcribed as 1400-nucleotide mRNA in vegetatively growing cells and can code for a 226 amino acid polypeptide (pdh1p) . Computational structural prediction has revealed that the pdh1p is a highly hydrophobic protein with seven transmembrane domains . The prediction has also detected a possible C-kinase phosphorylation site within the longest hydrophilic loop.

DNA Res, 1997 Dec 31, 4(6), 363 - 9
Identification of open reading frames in Schizosaccharomyces pombe cDNAs; Yoshioka S et al.; A total of 214 non-overlapping cDNA clones from Schizosaccharomyces pombe were selected and completely sequenced . The clones not previously reported were divided into the following three groups: 1) homologous to Saccharomyces cerevisiae genes (139 clones); 2) homologous to genes from other organisms but not to those from Sac . cerevisiae (4 clones); and 3) no similar sequences (40 clones) . Among the 31 sequences identical to those in the public databases, 4 genes have regions corresponding to introns . Protein sequences which had homologs both in budding yeast and mammals were compared with those from Sac . cerevisiae and mammals . The search revealed that the evolutionary distances among these species are similar at least with genes of this category.

J Biol Chem, 1998 Feb 20, 273(8), 4681 - 8
Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe; Jiang F et al.; The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis . Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe . In this work, we report the purification to homogeneity of PGPS from S . pombe . The enzyme was solubilized from the mitochondrial membrane of S . pombe with Triton X-100 . The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration . The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies . The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions . The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE . The enzyme showed a strong dependence on lipid cofactors for activity in vitro . While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin . The possible physiological significance of the lipid cofactor effect is discussed . This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S . pombe.

J Biol Chem, 1998 Feb 20, 273(8), 4666 - 71
A mammalian homolog of fission yeast Cdc5 regulates G2 progression and mitotic entry; Bernstein HS et al.; Progression through G2/M of the mammalian cell division cycle requires the coordinated expression of many gene products, but little is known of the transcriptional regulators involved . Schizosaccharomyces pombe Cdc5 is a putative transcription factor implicated in G2/M transit . We recently identified a cDNA encoding a putative human transcription factor, now designated human Cdc5 (hCdc5), with homology to S . pombe Cdc5 . Widespread expression of hCdc5 in human tissues and homology with expressed sequences in other eukaryotes suggested an evolutionarily conserved general function . Nuclear import of hCdc5 upon serum stimulation of mammalian cells suggested a possible role in cell proliferation . We now report that overexpression of hCdc5 in mammalian cells shortened G2 and reduced cell size . A dominant negative mutant of hCdc5 lacking the carboxyl-terminal activation domain slowed G2 progression and delayed entry into mitosis . Thus, hCdc5 is the first transcriptional regulator shown to affect G2 progression and mitotic entry in mammalian cells.

Hum Mol Genet, 1998 Feb, 7(2), 279 - 84
A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast; Yoder JA et al.; Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1 , the gene for the one previously recognized metazoan DNA methyltransferase . This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse . Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases . Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1 . Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation . The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids . Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.

J Cell Sci, 1998 Jan, 111 ( Pt 2), 149 - 59
Genes that cause aberrant cell morphology by overexpression in fission yeast: a role of a small GTP-binding protein Rho2 in cell morphogenesis; Hirata D et al.; To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression . The isolated genes were classified on the basis of morphology conferred . One of the genes causing a rounded morphology was identified as the rho2+ gene encoding a small GTP-binding protein . The overexpression of rho2+ resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall . Analyses using cdc mutants suggested that the overexpression of rho2+ prevents the establishment of growth polarity in G1 . The rho2+ gene was not essential, but among cells deleted for rho2+, those with an irregular shape were observed . The disruptant also showed a defect in cell wall integrity . An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment . A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site . The localization of GFP-Rho2 during interphase was partially dependent on sts5+ . These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction . Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.

Genetics, 1998 Feb, 148(2), 645 - 56
The identification of cDNAs that affect the mitosis-to-interphase transition in Schizosaccharomyces pombe, including sbp1, which encodes a spi1p-GTP-binding protein; He X et al.; Perturbations of the spi1p GTPase system in fission yeast, caused by mutation or overexpression of several regulatory proteins, result in a unique terminal phenotype that includes condensed chromosomes, a wide medial septum, and a fragmented nuclear envelope . To identify potential regulators or targets of the spi1p GTPase system, a screen for cDNAs whose overexpression results in this terminal phenotype was conducted, and seven clones that represent three genes, named med1, med2, and med3 (mitotic exit defect), were identified . Their genetic interaction with the spi1p GTPase system was established by showing that the spi1p guanine nucleotide exchange factor mutant pim1-d1ts was hypersensitive to their overexpression . med1 encodes a homologue of the human Ran-binding protein, RanBP1, and has been renamed sbp1 (spi1-binding protein) . sbp1p binds to spi1p-GTP and costimulates the GTPase-activating protein (GAP)-catalyzed GTPase activity . Cells in which sbp1p is depleted or overproduced phenocopy cells in which the balance between spi1p-GTP and spi1p-GDP is perturbed by other means . Therefore, sbp1p mediates and/or regulates the essential functions of the spi1p GTPase system . med2 and med3 encode novel fission yeast proteins that, based on our phenotypic analyses, are likely to identify additional regulators or effectors of the spi1p GTPase system.

FEBS Lett, 1998 Feb 6, 422(3), 321 - 7
A negative regulatory function for the protein tyrosine phosphatase PTP2C revealed by reconstruction of platelet-derived growth factor receptor signalling in Schizosaccharomyces pombe; Arkinstall S et al.; We have exploited reconstitution in the fission yeast Schizosaccharomyces pombe to investigate how activation of phospholipase Cgamma (PLCgamma) by the platelet-derived growth factor-beta receptor (PDGFbetaR) is regulated by the SH2 domain-containing protein tyrosine phosphatase PTP2C (also known as SHP-2) . When co-expressed in S . pombe, PTP2C abolished PDGFbetaR autophosphorylation as well as its ability to phosphorylate and activate PLCgamma . Inhibition of PDGFbetaR signalling by PTP2C appears specific insofar that PTPIC, a close homologue of PTP2C, does not suppress activation of either PDGFbetaR or PLCgamma . Surprisingly, an inactive PTP2C mutant (C459S), which dephosphorylates neither PDGFbetaR nor PLCgamma, remains fully effective as an inhibitor of {3H}inositol phosphate generation indicating that negative regulation is at least in part independent of catalytic activity . This contrasts with PLCgamma activation by c-Src which, although blocked by active PTP2C, is not inhibited by the mutant PTP2C C459S . These observations indicate that in addition to a reported positive role relaying trophic signals, PTP2C can also exert a negative effect on the PDGFbetaR and its signalling to PLCgamma.

J Bacteriol, 1998 Mar, 180(5), 1342 - 5
Characterization of mutants devoid of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe: partial protection from heat shock and high-salt stress; Cansado J et al.; Exposure of cells of Schizosaccharomyces pombe to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose . We constructed S . pombe mutants lacking neutral trehalase activity by gene replacement at the newly defined ntp1+ locus . Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media . Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation of ntp1+ in response to both stresses.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 647 - 52
Cloning, differential regulation and tissue distribution of alternatively spliced isoforms of ADP-ribosylation-factor-dependent phospholipase D from rat liver; Katayama K et al.; An alternatively spliced isoform of ADP-ribosylation-factor-dependent phospholipase D (PLD1) was previously shown to occur in rat C6 cells {Yoshimura, Nakashima, Ohguchi, Sakai, Shinoda, Sakai and Nozawa (1996) Biochem . Biophys . Res . Commun . 225, 494-499} and human HeLa cells {Hammond, Jenco, Nakashima, Cadwallader, Gu, Cook, Nozawa, Prestwich, Frohman and Morris (1997) J . Biol . Chem . 272, 3860-3868} . However, its complete sequence and the enzymological difference between the two PLD1 isoforms were unclear . Here we report the cloning, complete sequence, enzymological properties and tissue distribution of each of the two alternatively spliced PLD1 isoforms, a and b, from rat liver . The major difference between the two isoforms was the deletion of 38 amino acids in the b isoform, but otherwise the two cDNA sequences were 99.9% identical . The a-isoform sequence was 91% identical with the a form of human PLD1, and the 38-amino-acid deletion in the b form occurred at the same site as in the b form of human PLD1 . Both of the rat PLD1 isoforms expressed in the fission yeast Schizosaccharomyces pombe were dependent on ADP-ribosylation factor 1 and phosphatidylinositol 4,5-bisphosphate . The a isoform was activated by RhoA in a synergistic manner with ADP-ribosylation factor 1, whereas the b isoform was less responsive to RhoA . Reverse transcription PCR showed that the b form was the predominant PLD1 isoform expressed in rat tissues . The b-form transcript occurred in various rat tissues, including lung, brain, liver, kidney, small intestine and colon, whereas the a-form transcript was only detectable in lung, heart and spleen . Both transcripts were hardly detectable in thymus, stomach, testis and muscle . Thus the two PLD1 isoforms were differently regulated and expressed in rat tissues.

Mol Cell Biol, 1998 Mar, 18(3), 1670 - 81
Isolation, characterization, and molecular cloning of a protein (Abp2) that binds to a Schizosaccharomyces pombe origin of replication (ars3002); Sanchez JP et al.; The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe . A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S . pombe ARS consensus sequence previously reported by Maundrell et al . (K . Maundrell, A . Hutchison, and S . Shall, EMBO J . 7:2203-2209, 1988) . Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S . pombe crude extracts that bind to the oligonucleotide and ars3002 . One of these proteins, ARS binding protein 1, was previously described (Abpl {Y . Murakami, J . A . Huberman, and J . Hurwitz, Proc . Natl . Acad . Sci . USA 93:502-507, 1996}) . In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described . Purified Abp2 has an apparent molecular mass of 75 kDa . Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence . The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli . The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2 . The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences . Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2 . Disruption of the abp2 gene showed that the gene is not essential for cell viability . However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology . The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa . The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.

Appl Microbiol Biotechnol, 1998 Jan, 49(1), 45 - 50
Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe; Smerdon GR et al.; Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase . Integrant strains of S . pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media . Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media . Lipase activity is higher in complete medium than in minimal medium . Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid.

Mol Plant Microbe Interact, 1998 Mar, 11(3), 167 - 76
Carbon allocation in ectomycorrhizas: identification and expression analysis of an Amanita muscaria monosaccharide transporter; Nehls U et al.; Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants . An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus . As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria . Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters . A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction . This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A . muscaria mycorrhizal cDNA library . The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter . The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system . AmMst1 was constitutively expressed in fungal hyphae under all growth conditions . Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold . We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.

Yeast, 1998 Jan 30, 14(2), 189 - 94
Characterization of a branched-chain amino-acid aminotransferase from Schizosaccharomyces pombe; Eden A et al.; The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched-chain amino-acid aminotransferases (BCAT) were isolated recently . These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene . We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene . The S . pombe protein shows 47-52% identity to other eukaryotic BCAT proteins isolated from S . cerevisiae, nematode, mouse and man . A genetic growth assay for BCAT activity was established using an S . cerevisiae strain disrupted in both BCAT isoenzymes . Consequently, the activity of the S . pombe BCAT was demonstrated by genetic and biochemical means . Possible applications of BCAT-encoding genes as selection markers in yeast transformation are proposed.

Genes Dev, 1998 Feb 1, 12(3), 382 - 95
S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe; Lindsay HD et al.; Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest . Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis . Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents . Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G1 or G2 . Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26 . Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response . We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins . This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins.

J Biol Chem, 1998 Jan 30, 273(5), 2624 - 30
A human homolog can functionally replace the yeast vesicle-associated SNARE Vti1p in two vesicle transport pathways; Fischer von Mollard G et al.; Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE) . Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions . Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi . Here we describe a human Vti1p homolog, hVti1 . Whereas vti1Delta cells are inviable, expression of hVti1 allows vti1Delta cells to grow at nearly the wild-type growth rate . When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi . Sequence comparisons with a Schizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted alpha-helix . Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins.

J Biol Chem, 1998 Jan 30, 273(5), 2583 - 90
Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay; Roy SK et al.; The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases . Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S . cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J . F . (1996) J . Biol . Chem . 271, 3398-3405) . However, this is quite unexpected, because S . cerevisiae does not have galactose residues in its glycoproteins . To address this question we have constructed S . cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase . The mnn1 mutant of S . cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase . We present here three lines of evidences for the existence of UDP-galactose transporter in S . cerevisiae . (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface . (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium . (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay . This transport in S . cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100 . The overall UDP-galactose transport in S . cerevisiae is comparable with that in S . pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S . pombe than in S . cerevisiae.

Biochem J, 1998 Jan 15, 329 ( Pt 2), 313 - 9
Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L; Maksel D et al.; The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described . Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced . The Ap4A hydrolase cDNA was cloned from L . angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence . The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da . When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM . These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1 . 17) . Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities . No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin.

Nucleic Acids Res, 1998 Jan 15, 26(2), 594 - 601
Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2; Oram M et al.; A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA junctions . The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four-way junction (with a fixed crossover point), a three-way junction, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre . The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue . These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae . A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database . The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1 . The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S . pombe cells . Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro . The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein.

Nucleic Acids Res, 1998 Jan 15, 26(2), 505 - 11
Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors; Gross T et al.; The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms . In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins . The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name . We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1 . This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members . The gene is not essential for growth . We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved . Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity . Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele . prp4 encodes a protein kinase involved in pre-mRNA splicing . These findings are consistent with the notion that Srp1 plays a role in the splicing process.

Nucleic Acids Res, 1997 Dec 15, 25(24), 5103 - 9
In vivo analyses of RNA polymerase I termination in Schizosaccharomyces pombe; Melekhovets YF et al.; Recent studies on the termination of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae and Schizosaccharomyces pombe have suggested a more complex mechanism then previously described in higher eukaryotes . Termination appears to occur when a DNA-bound Reb1 protein molecule induces polymerase to pause in the context of a release element {see Reeder,R.H . and Lang,W . (1994) Mol . Microbiol ., 12, 11-15} . Because these conclusions in yeast were based entirely on in vitro analyses, we have examined the same termination process in S.pombe by expressing targeted mutations in vivo . S1nuclease protection studies indicate three tandemly arranged termination sites with most transcripts very efficiently terminated at the first site, 267 nt after the 3' end of the mature 25S rRNA sequence . Termination at each site is mediated by conserved terminator elements which bear limited sequence homology with that of mouse and also can be identified in S.cerevisiae . Removal of the first terminator element transfers dominance to the second site and construction of a new single terminator element at +150 still results in efficient termination and rRNA processing without a need for an additional upstream element . Genomic 'footprint' analyses and gel retardation assays confirm a process mediated by a strongly interacting protein factor but implicate an alternate binding site . Removal of the 5' flanking sequence or structure also had no effect on the site or efficiency of termination . Taken together the results in vivo suggest that the termination process in this fission yeast more strongly resembles the single element-mediated mechanism initially reported in mouse and is not dependent on additional upstream sequence as first reported in S.cerevisiae and postulated to function in general.

Genetics, 1998 Jan, 148(1), 49 - 57
Isolation of a Schizosaccharomyces pombe rad21ts mutant that is aberrant in chromosome segregation, microtubule function, DNA repair and sensitive to hydroxyurea: possible involvement of Rad21 in ubiquitin-mediated proteolysis; Tatebayashi K et al.; The fission yeast DNA repair gene rad21+ is essential for cell growth . To investigate the function essential for cell proliferation, we have isolated a temperature-sensitive mutant of the rad21+ gene . The mutant, rad21-K1, showed abnormal mitosis at the nonpermissive temperature . Some cells contained abnormal nuclear structures, such as condensed chromosomes with short spindles, or chromosomes stretched or unequally separated by elongating spindles . Other cells exhibited the displaced nucleus or a cut-like phenotype . Similar abnormalities were observed when the Rad21 protein was depleted from cells . We therefore concluded that Rad21 is essential for proper segregation of chromosomes . Moreover, the rad21-K1 mutant is sensitive not only to UV and gamma-ray irradiation but to thiabendazole and hydroxyurea, indicating that Rad21 plays important roles in microtubule function, DNA repair, and S phase function . The relation to the microtubule function was further confirmed by the fact that rad21+ genetically interacts with tubulin genes, nda2+ and nda3+ . Finally, the growth of the rad21-K1 mutant was inhibited at the permissive temperature by introduction of another mutation in the cut9+ gene, coding for a component of the 20S cyclosome/anaphase promoting complex, which is involved in ubiquitin-mediated proteolysis . The results suggest that these diverse functions of Rad21 may be facilitated through ubiquitin-mediated proteolysis.

J Bacteriol, 1998 Feb, 180(4), 892 - 900
Coordination of initiation of nuclear division and initiation of cell division in Schizosaccharomyces pombe: genetic interactions of mutations; Grallert A et al.; sep1+ encodes a Schizosaccharomyces pombe homolog of the HNF-3/forkhead family of the tissue-specific and developmental gene regulators identified in higher eukaryotes . Its mutant allele sep1-1 causes a defect in cytokinesis and confers a mycelial morphology . Here we report on genetic interactions of sep1-1 with the M-phase initiation mutations wee1-, cdc2-1w, and cdc25-22 . The double mutants sep1-1 wee1- and sep1-1 cdc2-1w form dikaryon cells at high frequency, which is due to nuclear division in the absence of cell division . The dikaryosis is reversible and suppressible by cdc25-22 . We propose that the genes wee1+, cdc2+, cdc25+, and sep1+ form a regulatory link between the initiation of mitosis and the initiation of cell division.

Biochemistry, 1998 Jan 13, 37(2), 590 - 5
Substrate specificity of Schizosaccharomyces pombe Nth protein for products of oxidative DNA damage; Karahalil B et al.; A gene from Schizosaccharomyces pombe, which encodes a protein with a strong sequence similarity to the Nth protein of Escherichia coli, has recently been identified {Roldan-Arjona, T., Anselmino, C., and Lindahl, T . (1996) Nucleic Acids Res . 24, 3307-3312} . The functional analysis of this eukaryotic enzyme indicated that it is a homologue of E . coli Nth protein . The gene has been subcloned and the protein (Nth-Spo) purified to apparent homogeneity . We investigated the substrate specificity of this eukaryotic enzyme for modified bases in oxidatively damaged DNA, using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS) . DNA substrates containing up to 17 types of modified bases were prepared by gamma-irradiation or by treatment with H2O2 in the presence of Fe(III)-EDTA or Cu(II) . The results revealed an efficient excision of five pyrimidine-derived lesions, 5-hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil . None of the other pyrimidine or purine lesions was excised . Excision was measured as a function of enzyme concentration, time, substrate concentration, and temperature . Kinetic constants were determined . Although some DNA base lesions removed by Nth-Spo protein were similar to those previously described for E . coli Nth protein, differences between substrate specificities of these two enzymes were noted.

Genes Dev, 1998 Jan 1, 12(1), 84 - 94
Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p; Sohrmann M et al.; Schizosaccharomyces pombe divides by means of a centrally placed division septum . The initiation of septation must be tightly coordinated with events in mitosis, as premature formation of the septum can lethally cut the undivided nucleus . The Spg1p GTPase and the Cdc7p kinase, with which it interacts, play a central role in signaling the initiation of septum formation . Loss-of-function mutations in either gene prevent septation, whereas inappropriate activation of Spg1p can induce septum formation from G1 or G2 interphase cells . Increased expression of either gene leads to multiple rounds of septation without cell cleavage, emphasizing the need for precise cell cycle regulation of their activity . To understand the mechanisms underlying this regulation, we have investigated whether these key initiators of septum formation are controlled by changes in their activity and/or location during mitosis and cytokinesis . We demonstrate that Spg1p localizes to the spindle pole body in interphase and to both spindle poles during mitosis . In contrast, Cdc7p shows no discrete localization during interphase, but early in mitosis it associates with both spindle pole bodies and, as the spindle extends, is seen on only one pole of the spindle during anaphase B . Spg1p activity is required for localization of Cdc7p in vivo but not for its kinase activity in vitro . Staining with an antiserum that recognizes preferentially GDP-Spg1p indicates that activated GTP-Spg1p predominates during mitosis when Cdc7p is associated with the spindle pole body . Furthermore, staining with this antibody shows that asymmetric distribution of Cdc7p may be mediated by inactivation of Spg1p on one spindle pole . Deregulated septation in mutant cells correlates with segregation of Cdc7p to both spindle poles.

Cytogenet Cell Genet, 1997, 78(3-4), 275 - 80
Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P); Vanhoof G et al.; A novel human cDNA (XPNPEPL) encoding a protein of 623 amino acids exhibiting 44% sequence identity and 62% sequence similarity to pig kidney X-prolyl aminopeptidase (aminopeptidase P; EC 3.4.11.9) was obtained by reverse transcription/polymerase chain reaction of phytohemagglutinin-stimulated lymphocyte mRNA . Conserved sequences were found with the prokaryotic X-prolyl aminopeptidase encoding gene (pepP) . The human gene translation product exhibits a high sequence homology to the Schizosaccharomyces pombe chromosome I hypothetical protein C22G7.01c and to the S . cerevisiae ORF y11029w . Northern blot analysis indicates an ubiquitous expression of the human XPNPEPL sequence.

Appl Environ Microbiol, 1998 Feb, 64(2), 555 - 63
Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414; Okada H et al.; We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-beta-1,4-glucanase) gene from Trichoderma reesei QM9414 . The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide . The deduced protein sequence has significant homologies with family H endo-beta-1,4-glucanases . The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion . Northern blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by glucose . The protein has been expressed as a mature form in Escherichia coli and as secreted forms in Saccharomyces cerevisiae and Schizosaccharomyces pombe under the control of tac, alcohol dehydrogenase (ADH1), and human cytomegalovirus promoters, respectively . The S . cerevisiae and Schizosaccharomyces pombe recombinant strains showed strong cellulolytic activities on agar plates containing carboxymethyl cellulose . The E . coli strain expressed small amounts of EG III in an active form and large amounts of EG III in an inactive form . The molecular masses of the recombinant EG IIIs were estimated to be 25, 28, and 29 kDa for E . coli, S . cerevisiae, and Schizosaccharomyces pombe, respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis . Parts of the yeast recombinant EG IIIs decreased their molecular masses to 25 kDa after treatment with endoglycosidase H and alpha-mannosidase, suggesting that they are N glycosylated at least partly.

Cell Motil Cytoskeleton, 1997, 38(4), 385 - 96
Type II myosin involved in cytokinesis in the fission yeast, Schizosaccharomyces pombe; May KM et al.; We have cloned an unique gene encoding the heavy chain of a type II myosin in the fission yeast, Schizosaccharomyces pombe . The myo2+ gene encodes a protein of 1526 amino acids with a predicted molecular weight of 177 kDa and containing consensus binding motifs for both essential and regulatory light chains . The S . pombe myo2+ head domain is 45% identical to myosin IIs from Saccharomyces cerevisiae and Homo sapiens and 40% identical to Drosophila melanogaster Structurally, myo2+ most closely resembles budding yeast MYO1, the tails of both myosin IIs containing a number of proline residues that are predicted to substantially disrupt the ability of these myosins to form coiled coils . The myo2+ gene is located on chromosome III, 8.3 map units from ade6+ . Deletion of approximately 70% of the coding sequence of myo2+ is lethal but myo2delta spores can acquire a suppressor mutation that allows them to form viable microcolonies consisting of filaments of branched cells with aberrant septa . Overexpression of myo2+ results in the inhibition of cytokinesis; cells become elongated and multinucleate and fail to assemble a functional cytokinetic actin ring and are either aseptate or form aberrant septa . These results suggest that a contractile actin-myosin based cytokinetic mechanism appeared early in the evolution of eukaryotic cells and further emphasise the utility of fission yeast as a model organism in which to study the molecular and cellular basis of cytokinesis.

FEBS Lett, 1997 Dec 29, 420(2-3), 161 - 6
Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe; Motegi F et al.; We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain . myo3 null cells showed a defect in cytokinesis under certain conditions . Overproduction of Myo3 also showed a defect in cytokinesis . Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin . Myo3 may be implicated in cytokinesis and stabilization of F-actin cables . Moreover, the function of Myo2 can be replaced by overexpressed Myo3 . We observed a modest synthetic interaction between Myo2 and Myo3 . Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S . pombe.

J Biol Chem, 1998 Jan 16, 273(3), 1654 - 61
Trichothecene 3-O-acetyltransferase protects both the producing organism and transformed yeast from related mycotoxins . Cloning and characterization of Tri101; Kimura M et al.; Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms . The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system . The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol) . A gene responsible for the 3-O-acetylation reaction, Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed in Schizosaccharomyces pombe . Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101 . This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms . The open reading frame of Tri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far . TRI101 from recombinant Escherichia coli catalyzes O-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner . By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F . graminearum . Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones . These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13765 - 70
Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe; Kon N et al.; Homologous recombination hotspots increase the frequency of recombination in nearby DNA . The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5'-ATGACGT-3') defined by extensive mutagenesis . A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold . Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity . This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot . Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses . However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities . Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels . These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination . Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S . pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

J Cell Sci, 1997 Oct, 110 ( Pt 20), 2599 - 608
Onset of gluconate-H+ symport in Schizosaccharomyces pombe is regulated by the kinases Wis1 and Pka1, and requires the gti1+ gene product; Caspari T; In the fission yeast Schizosaccharomyces pombe, glucose represses onset of gluconate-H+ symport and inhibits transiently the activity of the symport protein . Wild-type cells harvested from high glucose medium take up gluconate very slowly and the rate of uptake is increased 150-fold in response to glucose starvation . Here it is shown that an intact cAMP cascade is necessary to prevent premature onset in the presence of high glucose concentrations . Cells which have lost either adenylate cyclase (Cyr1) or cAMP-dependent protein kinase (Pka1) transport gluconate up to 60-fold faster than wild-type cells when harvested from high glucose medium . Moreover, inactivation of the stress-sensing Wis1-Sty1 MAP kinase pathway, by loss of Wis1 MAP kinase kinase, diminishes 10-fold the onset of gluconate uptake in response to starvation . A mutant was identified showing a comparable phenotype . By complementation, the gti1+ (gluconate transport inducer 1) gene has been isolated . Disruption of gti1 reduces starvation-induced onset by a similar factor to that observed in wis1 delta cells . Cells over-expressing gti1+ induce gluconate uptake much faster resulting in a threefold higher uptake rate, although gti1+ does not code for the gluconate transport protein . In contrast to the repression of onset, transient downregulation of the gluconate symporter is independent of Pka1 activity and requires ongoing glucose influx . Addition of glucose to starved cyr1 delta cells reduces uptake 9-fold, whereas starved pka1 delta cells, which are able to synthesise cAMP, respond with a 60-fold decrease in transport.

J Cell Sci, 1997 Oct, 110 ( Pt 20), 2547 - 55
Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton; Arellano M et al.; The Schizosaccharomyces pombe rho1p GTPase directly activates the (1-3) beta-D-glucan synthase and participates in the regulation of cell wall growth and morphogenesis in this fission yeast . Indirect immunofluorescence experiments using rho1p tagged with hemagglutinin have revealed that rho1p was located at the growing tips during interphase and at the septum prior to cytokinesis, localising to the same areas as actin patches . In S . pombe cdc10-129 mutant cells, arrested in G1, HA-rho1p accumulates at one tip whereas in cdc25-22 mutants, arrested in G2, HA-rho1p accumulates at both tips . In tea1-1 and tea2-1 cdc11-119 mutant cells, HA-rho1p is localised to the new growing tips . Overexpression of different rho1 mutant alleles caused different effects on cortical actin patch distribution, (1-3) beta-D-glucan synthase activation, and sensitivity to cell wall specific antifungal drugs . These results indicate that multiple cellular components are activated by rho1p . Overexpression of the dominant negative rho1T20N allele was lethal as was the rho1+ deletion . Moreover, when rho1+ expression was repressed in actively growing S . pombe, cells died in about 10 to 12 hours . Under these conditions, normal cell morphology was maintained but the level of (1-3) beta-D-glucan synthase activity decreased and the actin patches disappeared . Most cells lysed after cytokinesis during the process of separation, and lysis was not prevented by an osmotic stabiliser . We conclude that rho1p localisation is restricted to growth areas and regulated during the cell cycle and that rho1p is involved in cell wall growth and actin cytoskeleton organisation in S . pombe.

J Mol Biol, 1997 Nov 7, 273(4), 782 - 8
Interdependence in the processing of ribosomal RNAs in Schizosaccharomyces pombe; Good L et al.; Eukaryotic rRNAs are produced by cleavage of a large 35 to 45 S pre-rRNA transcript which initially must be fully transcribed and assembled into an 80 to 90 S nucleolar ribonucleoprotein particle . Despite this need for a completed transcript, several investigations have reported a split processing scheme for independent maturation of the large and small subunit rRNAs . Here, an efficiently expressed rDNA plasmid was used to quantitatively analyze the effects of mutations in the internal transcribed spacer (ITS) region in the yeast, Schizosaccharomyces pombe . The results show that substitution of ITS regions inhibits the processing of distant external transcribed spacers (ETS) and that deletion of the ITS2 spacer not only prevents the maturation of the large subunit, but severely affects maturation of the small subunit rRNA . This indicates that the processing mechanisms are not fully split and, when taken together with other evidence of interdependences in rRNA maturation, the results suggest that the interdependences act as a quality control mechanism to help ensure that only functional rRNA is incorporated into ribosomes.

Nucleic Acids Res, 1997 Nov 15, 25(22), 4700 - 1
Isolation of nuclei for chromatin analysis in fission yeast; Mason JA et al.; The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin . Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.

Nucleic Acids Res, 1997 Nov 15, 25(22), 4658 - 65
Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast; Romfo CM et al.; While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention . In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron . We have investigated the contribution of these two sequence elements to splicing in S.pombe . To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site . Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals . To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs . These changes block the first transesterification reaction, as in a subset of mammalian introns . However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract . Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns . Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA . These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.

J Bacteriol, 1998 Feb, 180(3), 674 - 9
Std1, a gene involved in glucose transport in Schizosaccharomyces pombe; Mehta SV et al.; A wild-type strain, Sp972 h-, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated . Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal . They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective) . The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain . We confirmed the transport deficiency of these mutants by {14C}glucose uptake . They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain . Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S . pombe, unlike in Saccharomyces cerevisiae . Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose . This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose . The lag disappears when the culture is transferred from the log phase of its growth on high concentrations . These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617-2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains . These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.

Exp Cell Res, 1998 Jan 10, 238(1), 220 - 30
Ribosomal DNA replication in the fission yeast, Schizosaccharomyces pombe; Sanchez JA et al.; We have employed genetic and two-dimensional (2D) gel electrophoretic methods to identify replication initiation, pausing, and termination sites in the tandem ribosomal DNA (rDNA) repeats of the fission yeast, Schizosaccharomyces pombe . An autonomously replicating sequence (ARS) element, ars3001, maps to a 2.3-kb restriction fragment spanning the junction between the nontranscribed spacer (NTS) and the external transcribed spacer upstream of the ribosomal RNA genes, and 2D gel analysis shows that replication initiates in the NTS portion of the same fragment . A pause region at the 3' end of the rRNA genes inhibits forks from entering these genes counter to the direction of transcription . Thus, most forks move through the genes in the same direction as transcription . In these respects, fission yeast rDNA replication resembles that in the budding yeast, Saccharomyces cerevisiae, and in multicellular eukaryotic organisms . A feature which, so far, has been detected only in fission yeast is the pausing of replication forks in a broad region near the 5.8S rRNA gene.

Glycobiology, 1997 Dec, 7(8), 1181 - 91
Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase; Scocca JR et al.; A key step in the assembly of oligosaccharide-lipid intermediates in N-linked glycosylation is the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT) . Comparison of the amino acid sequences of L-G1PT from five diverse species showed 75 amino acids identical in all five proteins . Using site-directed mutagenesis, we analyzed the importance of a number of these conserved residues to the enzymatic activity of L-G1PT using a plasmid shuffling procedure in Schizosaccharomyces pombe . S . pombe cells containing a chromosomal deletion of the essential gpt+ gene are rescued by a plasmid containing the S . pombe gpt open reading frame . Replacement of that plasmid by a plasmid encoding a mutated hamster L-G1PT cDNA sequence indicated that the mutated protein provided sufficient enzyme activity to permit cell growth . Mutations of aspartic acid 252 and asparagine 185 did not allow plasmid shuffling, indicating these residues were essential for activity . A combination of mutations at asparagine 182 and tryptophan 122 did not allow plasmid shuffling, although the single mutations did . Overexpression of the mutant proteins in S . pombe conferred tunicamycin (TM) resistance, indicating that the mutant proteins had a conformation necessary for binding TM, a substrate analog . The mutant proteins were also detected in Western blots and were correctly localized to the membrane fractions . However, the overexpressed proteins did not increase the endogenous level of enzymatic activity in these cells, indicating they were enzymatically inactive.

Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14320 - 5
Identification and characterization of a human protein kinase related to budding yeast Cdc7p; Jiang W et al.; The Cdc7p protein kinase is essential for the G1/S transition and initiation of DNA replication during the cell division cycle in Saccharomyces cerevisiae . Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from Schizosaccharomyces pombe . Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1 . The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa . HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three "kinase inserts" that are characteristic of Cdc7p and Hsk1 . Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1 in vitro . Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus . Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2 . These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G1/S phase transition and/or DNA replication in mammalian cells.

Genetics, 1997 Nov, 147(3), 1043 - 51
Mcs4, a two-component system response regulator homologue, regulates the Schizosaccharomyces pombe cell cycle control; Cottarel G; The Schizosaccharomyces pombe cdc2-3w wee1-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe . Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2 . Mutations in mcs1-6 suppress the cdc2-3w wee1-50 temperature-sensitive growth defect . Here, the cloning of mcs4 is described . The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family . Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium . These phenotypes are suppressed by a mutation in wee1 . In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen . These findings link the two-component signal transduction system to stress response and cell cycle control in S . pombe.

Genetics, 1997 Nov, 147(3), 1025 - 41
Mutational analysis of Cdc19p, a Schizosaccharomyces pombe MCM protein; Forsburg SL et al.; The cdc19+ gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe . We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches . We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p . We have found genetic interactions between cdc19+ and genes encoding subunits of DNA polymerase delta and the replication initiator cdc18+ . We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein . Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

FEBS Lett, 1997 Dec 22, 420(1), 39 - 42
A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe; Lafuente MJ et al.; We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli . These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ . The plasmids were constructed with the ura4+ or the his3+ marker of S . pombe . Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6-bisphosphatase and beta-galactosidase under the control of the fbp1+ promoter in different conditions.

Mol Cell Biol, 1998 Feb, 18(2), 953 - 9
Viral repression of fungal pheromone precursor gene expression; Zhang L et al.; Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus . This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture . At the molecular level, the virus is known to affect the transcription of a number of fungal genes . Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains . We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2 . To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains . The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus . Although published reports suggest that a G alpha(i) subunit may be involved in this regulation, our results do not support this hypothesis . The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe . This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete.

Mol Cell Biol, 1998 Feb, 18(2), 887 - 95
Novel factor highly conserved among eukaryotes controls sexual development in fission yeast; Okazaki N et al.; In the fission yeast Schizosaccharomyces pombe, the onset of sexual development is controlled mainly by two external signals, nutrient starvation and mating pheromone availability . We have isolated a novel gene named rcd1+ as a key factor required for nitrogen starvation-induced sexual development . rcd1+ encodes a 283-amino-acid protein with no particular motifs . However, genes highly homologous to rcd1+ (encoding amino acids with >70% identity) are present at least in budding yeasts, plants, nematodes, and humans . Cells with rcd1+ deleted are sterile if sexual development is induced by nitrogen starvation but fertile if it is induced by glucose starvation . This results largely from a defect in nitrogen starvation-invoked induction of ste11+, a key transcriptional factor gene required for the onset of sexual development . The striking conservation of the gene throughout eukaryotes may suggest the presence of an evolutionarily conserved differentiation controlling system.

J Virol, 1998 Feb, 72(2), 1324 - 33
The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration; Atwood A et al.; Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells . Schizosaccharomyces pombe is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics . In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition . In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration . By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts . These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition . Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain . These results suggested that each of the three IN domains was required for Tf1 transposition . The potential role of these five amino acid residues in the function of IN is discussed . Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase . The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis . These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts.

Annu Rev Genet, 1997, 31, 245 - 76
Mating type in filamentous fungi; Kronstad JW et al.; Mating type genes regulate sexual compatibility and sexual reproduction in fungi . This review focuses on recent molecular analyses of well-characterized mating systems from representative ascomycete (Neurospora crassa, Podospora anserina) and basidiomycete (Ustilago maydis, Coprinus cinereus, Schizophyllum commune) fungi . These mating systems include many conserved components, such as gene regulatory polypeptides and pheromone/receptor signal transduction cascades, as well as conserved processes, like self-nonself recognition and controlled nuclear migration . The components' structures and their genetic arrangements in the mating system vary greatly in different fungi . Although similar components and processes are also found in ascomycete yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe), the filamentous systems exhibit properties not encountered in yeast . Mating type genes act within, and control the development of, spatially differentiated fruiting bodies . The complex mating systems of basidiomycetes, unlike ascomycete systems, involve novel one-to-many specificity in both pheromone-receptor and homeodomain protein interactions.

Antonie Van Leeuwenhoek, 1997 Nov, 72(4), 327 - 35
The value of lipid composition in the taxonomy of the Schizosaccharomycetales; Jeffery J et al.; In this study, the lipid fractions i.e . neutral (NL), phospho-(PL) and glycolipids (GL) with associated fatty acids (FAs) of 54 strains, representing the Schizosaccharomycetales, were analyzed during stationary growth phase and compared . Trace amounts of linoleic acid (18:2) were present in most of the strains representing Schizosaccharomyces . An increased percentage 18:2 was observed in the PL fraction when compared to the NL fraction . This is possibly related to membranes requiring polyunsaturated FAs for fluidity . On the basis of the percentage oleic acid (18:1) and 18:2 FAs in the different lipid fractions, the Schizosaccharomycetales can clearly be divided into two groups i.e . Group 1 (represented by the genus Hasegawaea) comprising strains producing relatively large amounts of 18:2 and relatively low amounts of 18:1 when compared to Group 2 (represented by the genus Schizosaccharomyces comprising Schizosaccharomyces octosporus and Schizosaccharomyces pombe) . These results are in accordance with 18S and 26S rRNA base sequence analyses and emphasize the difference between the genera Hasegawaea and Schizosaccharomyces . Utilizing gas chromatography-mass spectrometry analyses, it was found that these strains were all capable of producing gamma-linolenic acid . This further emphasizes the uniqueness of this order in the Dikaryomycota.

Methods, 1997 Nov, 13(3), 221 - 33
Identification of autonomously replicating sequence (ARS) elements in eukaryotic cells; Clyne RK et al.; Autonomously replicating sequence (ARS) elements were first identified in the budding yeast Saccharomyces cerevisiae as chromosomal DNA fragments that promoted high frequency of transformation and extrachromosomal maintenance of plasmid DNA . These specific sequence elements were subsequently shown to function as origins of DNA replication . Detailed analysis of the structure and function of ARS elements has been limited largely to S . cerevisiae and more recently the fission yeast Schizosaccharomyces pombe . Characterization of ARS activity in other eukaryotes is far less complete . Here we describe the ARS assay developed in yeast and its application to the study of origin function in other eukaryotes . Other available methods for detecting autonomous replication in these systems are also presented.

Dev Biol, 1997 Dec 15, 192(2), 509 - 22
Imaginal tissues of Drosophila melanogaster exhibit different modes of cell proliferation control; Kylsten P et al.; The highly conserved regulatory mechanisms that control progression of a cell through the cell cycle do not, alone, explain the programmed control of cell proliferation during animal development . Additional controls must coordinate the cell cycle regulators with developmental regulatory events . Here we report studies of cell cycle control in the imaginal tissues of Drosophila melanogaster, specifically in situations where cell cycle progression is regulated by varying the length of the G2 phase . We show that G2-phase arrest in late larval wing imaginal disks requires transcriptional control of stg, a mitotic inducer that encodes a D . melanogaster homologue of the Schizosaccharomyces pombe p80cdc25 phosphatase . In a second study, string transcriptional regulation was also shown to be important for G2-phase regulation in eye disk cells posterior to the morphogenetic furrow . Finally, unlike all other situations described to date, string transcriptional regulation was found not to be the cause of G2 arrest in abdominal histoblasts, these cells being refractory to ectopic expression of stg . This study further establishes string as an important regulator of G2 phase during D . melanogaster development, but also reveals that at least one additional mechanism is utilized to control G2-phase length and thus cell proliferation in different developmental contexts.

J Biol Chem, 1998 Jan 16, 273(3), 1640 - 6
P-type ATPases mediate sodium and potassium effluxes in Schwanniomyces occidentalis; Banuelos MA et al.; Two genes isolated from Schwanniomyces occidentalis, ENA1 and ENA2, encode P-type ATPases highly homologous to the Na-ATPases of Saccharomyces cerevisiae and complement the Na+ sensitivity of an S . cerevisiae mutant strain lacking its own Na-ATPases . The expression of both ENA1 and ENA2 was highly dependent on a high external pH, but whereas a high pH was sufficient for the expression of ENA2, the expression of ENA1 required a high pH and the presence of Na+ . Disruption of ENA1 rendered the cells less tolerant to Na+ than the wild-type strain and decreased their capacity for Na+ extrusion . Disruption of ENA2 did not affect Na+ tolerance, but decreased both the growth at high pH and K+ efflux . We discuss these results and propose that fungal Na-ATPases should be considered alkali cation ATPases . By sequence comparison, we found that fungal Na-ATPases form a homogeneous group that can be distinguished from other cation-pumping P-type ATPases, except from the cta3 Ca-ATPase of Schizosaccharomyces pombe.

Toxicol Appl Pharmacol, 1997 Dec, 147(2), 312 - 8
Molecular mechanisms controlling sensitivity to toxic metal ions in yeast; Perego P et al.; Contamination of the environment has made toxic metal ions a major health issue . The use of yeasts as model systems for the identification of molecular mechanisms that control sensitivity to these agents is particularly attractive because of the ease of genetic manipulation and the availability of the complete Saccharomyces cerevisiae genomic sequence . This paper reviews information on those genes and mechanisms that have been identified in both the budding yeast S . cerevisiae and the fission yeast Schizosaccharomyces pombe as being capable of modulating sensitivity to important toxic metals . The factors that influence sensitivity to toxic metal ions include cellular thiols (glutathione, phytochelatins, labile sulfide, and metallothioneins) and the products of genes directly and indirectly involved in the transport or sequestration of the metal ion . A complete understanding of the molecular basis of sensitivity to toxic metal ions in lower organisms is expected to provide useful insights in the metal ion detoxification pathways and diseases related to these pathways in humans.

Can J Microbiol, 1997 Nov, 43(11), 991 - 8
Polarity, spatial organisation of cytoskeleton, and nuclear division in morphologically altered cells of Schizosaccharomyces pombe; Sipiczki M et al.; To gain more information about the determination of cell polarity and its relationship to the organisation of cytoskeleton, we have examined the mycelial mutant sep1-1 and the multinucleate multipolar syncytia of the triple mutant sep1-1 spl1-1 cdc4-8 by indirect immunofluorescence techniques . We have found that polarity is predetermined by the shape of the cell . During transition from mitosis to interphase the microtubules of the arising cytoplasmic cytoskeleton gradually form a basket-like pattern that reflects the curvatures of the cell envelope . The presumable growing poles, where actin accumulates, usually correlates with sites where the cell tapers and the microtubules converge . However, no growth can be launched at these sites if the cell surface has not been properly processed . Mitosis and meiosis are not affected significantly by changes in cell morphology and polarity, but larger cells are less effective during sporulation . The azygotic asci produced by multinucleate syncytia frequently contain over 20 ascospores.

Mol Gen Genet, 1997 Nov, 256(6), 638 - 51
G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair; Lydall D et al.; We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae . Mec3p shows no strong similarity to other proteins currently in the database . Rad17p is similar to Rec1 from Ustilago maydis, a 3' to 5' DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported) . Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S . pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair . This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA pol alpha (cdc17) and DNA pol delta (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC4 strains . The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC4 strains . Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage . In addition, all three are required for the rapid death of cdc13 rad9 mutants . All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest.

Eur J Biochem, 1997 Dec 1, 250(2), 476 - 83
Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase; Balcells L et al.; In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases . On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene . We have isolated a S . pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products . Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p . When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S . cerevisiae ppz1delta mutants . Disruption of pzh1 yields viable S . pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+ . Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism . Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.

Gene, 1997 Nov 20, 202(1-2), 1 - 5
sep1+ encodes a transcription-factor homologue of the HNF-3/forkhead DNA-binding-domain family in Schizosaccharomyces pombe; Ribar B et al.; We report on the cloning of sep1+, a gene whose mutation causes filamentous growth in Schizosaccharomyces pombe . Since cell growth and propagation are not affected by the mutation, it could not be cloned using selective conditions for the identification of the positive transformants . Instead, we cloned it from a cosmid of a contig (Hoheisel et al., Cell 73, 109-1120, 1993) supposed to cover the chromosomal region where the sep1-1 mutation mapped . The 1761 bp long ORF codes for a protein containing a sequence similar to the DNA-binding domains of the HNF-3/forkhead family of transcription factors.

J Cell Sci, 1997 Nov, 110 ( Pt 21), 2715 - 27
Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery; Broughton J et al.; The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit . In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved . In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica . The S . pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum . Sequence comparisons reveal that S . pombe Sec61p is 58.6% identical to that of S . cerevisiae . The deduced amino acid sequence of the Y . lipolytica protein shares 68.8% sequence identity with S . cerevisiae Sec61p . Gene disruption studies have shown that the SEC61 is required for viability in both S . pombe and Y . lipolytica demonstrating that the essential nature of this protein is not unique to S . cerevisiae . Moreover, heterologous complementation studies indicate that the Y . lipolytica SEC61 gene can complement a null mutation in S . cerevisiae . Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops . These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.

Mol Cell Biol, 1998 Jan, 18(1), 400 - 8
Activation of the kexin from Schizosaccharomyces pombe requires internal cleavage of its initially cleaved prosequence; Powner D et al.; Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway . The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe . Using a cell-free translation-translocation system prepared from Xenopus eggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum . The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain . Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102/ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site . Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans . Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars . O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site . Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme . Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence . Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S . pombe cells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.

Genomics, 1997 Dec 1, 46(2), 294 - 8
Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22; Ishiai M et al.; Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing . We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p . Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S . pombe, and S . cerevisiae Orc5p, respectively . We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization . We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1 . We have identified the S . pombe ORC5 gene and located it in a cosmid mapped on chromosome II.

J Cell Biol, 1997 Dec 1, 139(5), 1063 - 76
KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion; Beh CT et al.; KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating . To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product . KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei . KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion . Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion . Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum . In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy . We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.

Nature, 1997 Dec 18-25, 390(6661), 698 - 701
Identification and role of adenylyl cyclase in auxin signalling in higher plants; Ichikawa T et al.; Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes, but its significance in higher plants has been generally doubted because they have low adenylyl cyclase activity and barely detectable amounts of cAMP . Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media . The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant . Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe . Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation . Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide . This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

Gene, 1997 Nov 12, 201(1-2), 169 - 77
The mUBC9 murine ubiquitin conjugating enzyme interacts with the E2A transcription factors; Loveys DA et al.; The ubiquitin-mediated degradation of cellular proteins requires the sequential activity of E1, E2 and, in some cases, E3 enzymes . Using the yeast two-hybrid system, we have cloned 1.0- and 2.5-kb cDNAs encoding the identical murine E2, or ubiquitin conjugating enzyme by virtue of its interaction with the E2A transcription factor . This cDNA encodes the 158-amino-acid protein, mUBC9, which has considerable sequence homology to UBC9 from Saccharomyces cerevisiae and HUS5 from Schizosaccharomyces pombe and is identical to the human UBC9 protein . HUS5 is essential for DNA damage repair, whereas UBC9 is necessary for G2/M progression . The human protein has been shown to correct the UBC9 defect in yeast . Antisera raised against bacterially expressed mUBC9 fusion protein recognize a murine cellular protein of approximately 18 kDa, corresponding to the predicted mobility . Unlike E2A, the mUBC9 protein level is not regulated by serum growth factors . The activity of the apparent homologues UBC9 and HUS5 suggests that mUBC9 may be involved in the degradation of key nuclear proteins that regulate cell cycle progression.

J Biol Chem, 1997 Nov 28, 272(48), 30470 - 5
Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit; Spataro V et al.; We have investigated the usefulness of the fission yeast Schizosaccharomyces pombe as a model organism for the discovery of novel modes of drug resistance in human cells . In fission yeast, overexpression of the essential pad1(+) gene confers pleiotropic drug resistance through a pathway involving an AP-1 transcription factor encoded by pap1(+) . We have identified POH1, a human pad1 homologue that can substitute fully for pad1(+) and induce AP-1-dependent drug resistance in fission yeast . POH1 also confers P-glycoprotein-independent resistance to taxol (paclitaxel), doxorubicin, 7-hydroxystaurosporine, and ultraviolet light when transiently overexpressed in mammalian cells . Poh1 is a previously unidentified component of the human 26 S proteasome, a multiprotein complex that degrades proteins targeted for destruction by the ubiquitin pathway . Hence, Poh1 is part of a conserved mechanism that determines cellular susceptibility to cytotoxic agents, perhaps by influencing the ubiquitin-dependent proteolysis of transcription factors.

Gene, 1997 Oct 24, 200(1-2), 135 - 44
Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins; Lu Q et al.; We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag . This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism . Unlike E . coli, S . pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins . Two vectors have been constructed for protein expression in S . pombe, pESP-1 and pESP-2 . Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest . Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads . The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease . Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase . Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.

Gene, 1997 Oct 24, 200(1-2), 11 - 23
Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus; Tobin MB et al.; Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus . In A . fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified . One gene (AflMDR1) was isolated from A . flavus and is the apparent homologue to AfuMDR1 . AfuMDR1 and AflMDR1 encode proteins of molecular weights 148,000 and 143,000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites . These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site . The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1 . The second gene identified in A . fumigatus, AfuMDR2, encodes a protein of molecular weight 85,000, containing four putative transmembrane domains and an ATP binding domain . The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae . Expression of AFUMDR1 in S . cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.

Mol Cell Biol, 1997 Dec, 17(12), 7047 - 60
Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export; Yoon JH et al.; To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167 . Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa) . This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae . Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery . In contrast to NIC96, the npp106 gene is not essential . Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus . A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions . In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106 . We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality . These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.

Mol Cell Biol, 1997 Dec, 17(12), 7008 - 18
The Saccharomyces cerevisiae Hap5p homolog from fission yeast reveals two conserved domains that are essential for assembly of heterotetrameric CCAAT-binding factor; McNabb DS et al.; The CCAAT-binding factor is an evolutionarily conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites within the promoters of numerous eukaryotic genes . The CCAAT-binding factor from Saccharomyces cerevisiae is a heterotetramer that contains the subunits Hap2p, Hap3p, Hap4p, and Hap5p and that functions in the activation of genes involved in respiratory metabolism . Here we describe the isolation of the cDNA encoding the Schizosaccharomyces pombe homolog of Hap5p, designated php5+ . We have shown that Php5p is a subunit of the CCAAT-binding factor in fission yeast and is required for transcription of the S . pombe cyc1+ gene . Analysis of the evolutionarily conserved regions of Hap5p, Php5p, and the mammalian homolog CBF-C revealed two essential domains within Hap5p that are required for DNA binding and transcriptional activation . One is an 87-amino-acid core domain that is conserved among Hap5p, Php5p, and CBF-C and that is required for the assembly of the Hap2p-Hap3p-Hap5p heterotrimer both in vitro and in vivo . A second domain that is essential for the recruitment of Hap4p into the CCAAT-binding complex was identified in Hap5p and Php5p.

Mol Cell Biol, 1997 Dec, 17(12), 6868 - 75
Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance; Murray JM et al.; The cellular responses to DNA damage are complex and include direct DNA repair pathways that remove the damage and indirect damage responses which allow cells to survive DNA damage that has not been, or cannot be, removed . We have identified the gene mutated in the rad12.502 strain as a Schizosaccharomyces pombe recQ homolog . The same gene (designated rqh1) is also mutated in the hus2.22 mutant . We show that Rqhl is involved in a DNA damage survival mechanism which prevents cell death when UV-induced DNA damage cannot be removed . This pathway also requires the correct functioning of the recombination machinery and the six checkpoint rad gene products plus the Cdsl kinase . Our data suggest that Rqh1 operates during S phase as part of a mechanism which prevents DNA damage causing cell lethality . This process may involve the bypass of DNA damage sites by the replication fork . Finally, in contrast with the reported literature, we do not find that rqh1 (rad12) mutant cells are defective in UV dimer endonuclease activity.

Mutat Res, 1997 Oct, 385(1), 47 - 57
Molecular analysis of ERCC2 mutations in the repair deficient hamster mutants UVL-1 and V-H1; Kadkhodayan S et al.; The cDNA sequence of the Chinese hamster ERCC2 nucleotide excision repair and transcription gene from the UVL-1 Chinese hamster ovary (CHO) mutant cell line and the V-H1 Chinese hamster V79 mutant line was analyzed . ERCC2 encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the structural hemizygosity of chromosome 9 . Both UVL-1 and V-H1 have intermediate levels of (6-4) photoproduct repair but are as highly UV sensitive as the group 2 mutants that have no detectable repair . Deficiency in cyclobutane dimer removal has also been shown for V-H1 . In UVL-1, a single base substitution resulting in an Arg75-->Trp substitution in helicase domain Ia was identified . The equivalent amino acid position is also Arg in the human, mouse, Xiphophorus maculatus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe homologs . In V-H1, a single base substitution resulting in a Thr46-->Ile substitution in helicase domain I (the ATP-binding domain) was identified in both alleles . The equivalent amino acid position is also Thr in the five homologs . Analysis of three V-H1 partial revertants revealed that they still have the original V-H1 mutation in both alleles, indicating that these are second site reversion events . Site-specific mutagenesis was used to introduce the Thr46-->Ile, Arg75-->Trp, and Lys48-->Arg (helicase domain I) mutations into a hamster ERCC2 expression plasmid . These plasmids each failed to confer UV resistance to group 2 mutant cells, further demonstrating that the changes identified are the causative mutations in V-H1 and UVL-1 . Correlations between specific mutations, biochemical activities, and repair phenotype are discussed.

J Biol Chem, 1997 Nov 21, 272(47), 29742 - 51
Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins; Kudo N et al.; Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity . cDNA encoding a human homolog of Crm1 was cloned . Human CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S . pombe Crm1 . hCRM1 weakly complemented the cold-sensitive mutation of S . pombe crm1-809, as did S . pombe crm1+ . Overproduction of hCRM1 under the control of a series of nmt1 promoters suppressed cell proliferation in wild-type S . pombe in an expression level-dependent manner . A similar inhibitory effect was also observed for crm1+ . Cells overproducing either hCRM1 or S . pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei . Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa . Closely similar phenotypes between hCRM1- and S . pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S . pombe crm1+ . Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle . Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm . A crm1 mutation of S . pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal . All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.

RNA, 1997 Dec, 3(12), 1434 - 43
The La protein in Schizosaccharomyces pombe: a conserved yet dispensable phosphoprotein that functions in tRNA maturation; Van Horn DJ et al.; Most RNA polymerase III transcripts are bound immediately after synthesis by an abundant nuclear phosphoprotein known as the La autoantigen . Experiments performed in the budding yeast Saccharomyces cerevisiae have revealed that binding of the La protein to tRNA precursors is required for the endonucleolytic maturation of the 3' terminus of many tRNAs . In the absence of this protein, the 3' ends of these tRNAs are trimmed by exonucleases (Yoo CJ, Wolin SL, 1997, Cell 89:393-402) . Here we report the characterization of the La protein in the fission yeast Schizosaccharomyces pombe . As was described for budding yeast, S . pombe cells lacking the La protein are viable and exhibit alterations in the pathway of pre-tRNA maturation . Introduction of either the human, S . cerevisiae, or S . pombe La protein into these cells restores the detected pattern of tRNA processing intermediates to that of wild-type cells . By performing immunoprecipitations from cells that were metabolically labeled with 32P-orthophosphate, we demonstrate that the S . pombe and S . cerevisiae La proteins, like the human La protein, are phosphorylated in vivo . Thus, although the La protein is dispensable for growth in these yeasts, both the structure of the protein and its function in pre-tRNA maturation have been highly conserved throughout evolution.

J Bacteriol, 1997 Dec, 179(24), 7653 - 62
cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B; Ishiguro J et al.; The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J . Ishiguro and Y . Uhara, Jpn . J . Genet . 67:97-109, 1992) . We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C . Under any of these restrictive treatments, cells swell up and finally lyse . With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched . The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount) . Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes . All of these data suggest a relationship between the cps1+ gene and cell wall synthesis . A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains . S . pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase . Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis.

Mol Biol Cell, 1997 Dec, 8(12), 2693 - 705
Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis; Bezanilla M et al.; As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring . We cloned myp2(+), a myosin-II in Schizosaccharomyces pombe, conditionally required for cytokinesis . myp2(+), the second myosin-II identified in S . pombe, does not completely overlap in function with myo2(+) . The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family . The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs . However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines . Disruption of myp2(+) is not lethal but under nutrient limiting conditions cells lacking myp2(+) function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis . The presence of salt enhances these morphological defects . Additionally, Deltamyp2 cells are cold sensitive in high salt, failing to form colonies at 17 degrees C . Thus, myp2(+) is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth . GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis . Additionally, we constructed double mutants of Deltamyp2 with temperature-sensitive mutant strains defective in cytokinesis . We observed synthetic lethal interactions between Deltamyp2 and three alleles of cdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicating myp2(+) function for efficient cytokinesis under normal conditions.

Mol Biol Cell, 1997 Dec, 8(12), 2475 - 86
A WD repeat protein controls the cell cycle and differentiation by negatively regulating Cdc2/B-type cyclin complexes; Yamaguchi S et al.; In the fission yeast Schizosaccharomyces pombe, p34(cdc2) plays a central role controlling the cell cycle . We recently isolated a new gene named srw1(+), capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34(cdc2) . Cells lacking srw1(+) are sterile and defective in cell cycle controls . When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34(cdc2)/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase . Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34(cdc2) . srw1(+) shares functional similarity with rum1(+), having abilities to induce endoreplication and restore fertility to rum1 disruptants . In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen . We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34(cdc2) and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.

Nat Genet, 1997 Dec, 17(4), 498 - 502
Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT; Weinrich SL et al.; The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules . Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours . However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division . It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation . In addition to the human telomerase RNA component (hTR; ref . 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans . More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit . A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues . We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme . Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase . Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells . The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Can J Microbiol, 1997 Oct, 43(10), 945 - 53
Colocalization of microtubules and mitochondria in the yeast Schizosaccharomyces japonicus var . versatilis; Svoboda A et al.; Both living and fixed cells of Schizosaccharomyces japonicus var . versatilis showed thread-like mitochondria when studied by phase-contrast and fluorescence microscopy . In the interphase cells, mitochondria extended from pole to pole and converged towards the growing tips . The mitochondrial threads did not disrupt but persisted during mitosis and, subsequently, their bundle was split between the two daughter cells by a concentrically growing septum . Mitochondria in the interphase cells were accompanied by cytoplasmic microtubules . These disappeared during mitosis and, instead, spindle microtubules were formed in the nucleus . The cytoplasmic microtubules reappeared after anaphase B, again in coaligment with mitochondria . Protoplasting as well as the action of microtubule inhibitors methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) and 2-methylbenzimidazole (MBC) resulted in rapid disintegration of microtubules and, suprisingly, also in disruption of mitochondria into small bodies . Removal of the inhibitors or a short regeneration of protoplasts allowed both the cytoplasmic microtubules and the thread-like mitochondria to reaggregate into the original pattern . Cytochalasin D treatment caused a complete disintegration of actin filaments, while the cytoplasmic microtubules and mitochondria remained intact . These findings of a transient close association of mitochondria and microtubules and their relative independence of the arrangement of actin filaments suggest that microtubules, but not actin cables, form supports for positioning or movement of mitochondria along the cylindrical cells . The persistence of mitochondria in the cell centre during mitosis may be accounted for by the fact that disrupted microtubules fail to provide support for mitochondrial movement towards the cell poles.

Yeast, 1997 Nov, 13(14), 1329 - 35
Measurement of nuclear DNA content in fission yeast by flow cytometry; Carlson CR et al.; Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature . Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point . The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S . Sazer and S . W . Sherwood, J . Cell Sci . 97: 509-516, 1990) . Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis . To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion . The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size . With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content . Premature and abnormal mitosis ('cut') could be observed for the orp1 mutant after only 4 h at restrictive temperature.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 187 - 91
Phosphatidylglycerophosphate synthase from yeast; Minskoff SA et al.; The phospholipid cardiolipin, or diphosphatidylglycerol, is ubiquitous in eucaryotes . It is unique in structure, subcellular localization, and potential function . Because it is found predominantly in the mitochondrial inner membrane, it is an excellent marker for mitochondrial biogenesis . Cardiolipin is required for activity of several mitochondrial enzymes and possibly also for import of proteins into the mitochondrion . To understand the role of cardiolipin in these cellular events, it is necessary to characterize the enzymes of the cardiolipin pathway, as well as the genes that control the expression of these enzymes . To date, the structural genes encoding the cardiolipin biosynthetic enzymes have not been identified in any eucaryotic organism . However, considerable information is available regarding the regulation of this pathway in yeast . The activity and regulation of the first enzyme of the pathway, CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerophosphate (PGP) synthase, EC 2.7.8.5), has been characterized in two evolutionarily divergent yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe . In contrast to the second and third enzymes of the pathway, this enzyme is highly regulated, both by cross-pathway control and by factors affecting mitochondrial development . PGP synthase from S . pombe (and cardiolipin synthase from S . cerevisiae) have been purified to homogeneity . The amino acid sequences of these enzymes, combined with the availability of the complete genome sequence from S . cerevisiae will simplify the cloning of these genes in the near future.

Gene, 1997 Oct 1, 198(1-2), 251 - 8
The human RAE1 gene is a functional homologue of Schizosaccharomyces pombe rae1 gene involved in nuclear export of Poly(A)+ RNA; Bharathi A et al.; A Schizosaccharomyces pombe temperature-sensitive mutant, rae1-1, was previously identified by us as being defective in nuclear export of Poly(A)+ RNA when grown at restrictive temperature . Here, we report the isolation of the human homologue of the S . pombe rae1 gene . The RAE1 genes are highly conserved in evolution in both structure and function . The human RAE1 cDNA, when expressed from the CMV-promoter, can suppress partially the temperature sensitivity of the rae1-1 mutant . This is also reflected by increased Poly(A)+ RNA export at a restrictive temperature . An epitope tagged human Rae1p localizes to both the nucleus and the cytoplasm in transiently transfected HeLa cells . We discuss the potential role of Rae1p in nuclear cytoplasmic trafficking in yeast and higher eukaryotic cells.

Curr Biol, 1997 Oct 1, 7(10), 767 - 75
The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export; Neville M et al.; BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts . Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae . The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function . Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import . Crm1p has recently been shown to contribute to nuclear export in vertebrate systems . Here, we have studied this mechanism of nuclear to cytoplasmic transport . RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S . cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism . Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p . CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex . Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

Nature, 1997 Nov 13, 390(6656), 187 - 92
Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis; Dove SK et al.; Inositol phospholipids play multiple roles in cell signalling systems . Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3 . In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking . We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase . This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins . PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure . The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Genes Cells, 1997 Jul, 2(7), 467 - 79
A globular complex formation by Nda1 and the other five members of the MCM protein family in fission yeast; Adachi Y et al.; BACKGROUND: In the fission yeast Schizosaccharomyces pombe, Nda1, Nda4, Mis5 and Cdc21 proteins belong to the MCM (minichromosome maintenance) protein family which is thought to have six members . Each MCM member is required for the early stages of DNA replication, and has a well-conserved central 200-amino acid domain containing a putative ATP binding motif . However, the precise molecular functions of MCM proteins are not yet clear . RESULTS: We investigated the physical interaction of Nda1 protein with the other fission yeast MCM proteins using specific antibodies . Immunoprecipitation of Nda1 protein leads to the co-precipitation of all the other members of the fission yeast MCM protein family . We purified the MCM protein complex by a combination of column chromatography . The native molecular weight of the MCM complex was estimated by gel filtration to be 560 kDa . The purified fraction contained nearly equal quantities of the six MCM proteins . Electron microscope observation showed that the MCM complex has a globular shape with a central cavity . CONCLUSIONS: We have developed a procedure to purify fission yeast MCM proteins in a native hetero-oligomeric complex form for the first time, which opens an avenue to further biochemical analysis.

Biochim Biophys Acta, 1997 Oct 11, 1358(3), 229 - 39
Fission yeast dihydrolipoamide dehydrogenase gene is involved in G1/S cell cycle progression; Jang YJ et al.; Using functional complementation with a Schizosaccharomyces pombe genomic library, we have isolated a clone complementing a G1/S phase progression defective mutant . The newly isolated temperature-sensitive mutant, cyj150, showed elongated morphology at a restrictive temperature of 36 degrees C and DNA content analysis of the mutant indicated a defect in cell cycle progression at the G1/S phase . Sequence analysis of the genomic and cDNA clones complementing this elongated phenotype at 36 degrees C show that it encodes a protein that has 50% amino acid identity with dihydrolipoamide dehydrogenase from Saccharomyces cerevisiae and garden pea . Alignment of the deduced amino acid sequence of S . pombe dihydrolipoamide dehydrogenase (dld1+) with glutathione reductase and mercuric reductase revealed extensive homologies throughout the primary sequence and protein structure, and contained amino acid sequences of the active site region conserved from prokaryote to higher eukaryote . Gene disruption and tetrad analysis showed that dld1+ is an essential gene for cell viability . Northern analysis indicates that transcriptional expression of this gene is not fluctuated according to the cell cycle . However, it is certain that malfunction of this Dld1 protein blocks the progression of cell cycle from G1 to S phase . The sequence of the dld1+ gene is available in EMBL/GenBank under Accession Number L40360.

Eur J Biochem, 1997 Oct 1, 249(1), 98 - 106
Protein phosphatase beta, a putative type-2A protein phosphatase from the human malaria parasite Plasmodium falciparum; Li JL et al.; Protein phosphatases play a critical role in the regulation of the eukaryotic cell cycle and signal transduction . A putative protein serine/threonine phosphatase gene has been isolated from the human malaria parasite Plasmodium falciparum . The gene has an unusual intron that contains four repeats of 32 nucleotides and displays a high degree of size polymorphism among different strains of P . falciparum . The open reading frame reconstituted by removal of the intron encodes a protein of 466 amino acids with a predicted molecular mass of approximately 53.7 kDa . The encoded protein, termed protein phosphatase beta (PP-beta), is composed of two distinct domains . The C-terminal domain comprises 315 amino acids and exhibits a striking similarity to the catalytic subunits of the type-2A protein phosphatases . Database searches revealed that the catalytic domain has the highest similarity to Schizosaccharomyces pombe Ppa1 (58% identity and 73% similarity) . However, it contains a hydrophilic insert consisting of five amino acids . The N-terminal domain comprises 151 amino acid residues and exhibits several striking features, including high levels of charged amino acids and asparagine, and multiple consensus phosphorylation sites for a number of protein kinases . An overall structural comparison of PP-beta with other members of the protein phosphatase 2A group revealed that PP-beta is more closely related to Saccharomyces cerevisiae PPH22 . Southern blots of genomic DNA digests and chromosomal separations showed that PP-beta is a single-copy gene and is located on chromosome 9 . A 2800-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage (gametocytes) . The results indicate that PP-beta may be involved in sexual stage development.

Eur J Biochem, 1997 Oct 1, 249(1), 61 - 9
Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase--involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway; Kribii R et al.; Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway . A full-length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches . Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array . SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity) . The isolated cDNA has been assigned to the SQS1 gene product, SQS1 . RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots . Arabidopsis SQS1 isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain . However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302 . Labeling studies using {3H}farnesyl-P2 and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S . pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12491 - 6
Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint; D'Urso G et al.; In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication . Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication . Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step . In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor {Navas, T . A., Zhou, Z . & Elledge, S . J . (1995) Cell 80, 29-39} . We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis . In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost . We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

Biol Chem, 1997 Sep, 378(9), 963 - 73
DNA replication and order of cell cycle events: a role for protein isoprenylation?
Galli I, Uchiyama M, Wang TS.
When the aya1+ gene is mutated, Schizosaccharomyces pombe cells become unable to react appropriately to a delay in DNA replication . Instead of stalling the cell cycle to allow completion of DNA synthesis, they proceed unperturbed towards mitosis and attempt to segregate the still unreplicated chromosomes . As a result, the genetic material segregates unevenly and the nuclei assume a mitotic catastrophe phenotype, characterized by torn chromosomes (cut), anucleated cells and scattered chromosomes . Interestingly, the aya1 phenotype can be suppressed by overexpression of either the catalytic subunit of S . pombe DNA polymerase alpha or of a novel protein called hur1 +p . The latter bears significant homology to the core of the human Rab escort protein, which belongs to a family of factors necessary to the post-translational isoprenylation of proteins like Ras, Rab and lamin B . When isoprenylation is chemically inhibited with R-limonene (a monoterpene derived from orange rind), wild type S . pombe cells become insensitive to an S phase delay, in a manner strongly reminiscent of aya1 mutants . Moreover, overexpression of hur1 +p in wild type cells rescues the failing checkpoint function . We propose that there is a strong correlation between the aya1 phenotype, S-M phase checkpoint function, and isoprenylation events in fission yeast.

J Biol Chem, 1997 Oct 31, 272(44), 27980 - 6
Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers; Thomas M et al.; To probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II . A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation . Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II . S . cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected . Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit . The selected RNA was expressed in yeast cells under the control of a Pol III promoter . Yeast cells that expressed the anti-Pol II aptamer grew normally . However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II.

Hum Mol Genet, 1997 Nov, 6(12), 2117 - 26
Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome; Wang A et al.; Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome . U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome . U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins . We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively . Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p . Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe . Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP . Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p . Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins . These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat . The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p . Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading . Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin . Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction . These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.

RNA, 1997 Oct, 3(10), 1182 - 93
Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe; Rotondo G et al.; The Pac1 ribonuclease of Schizosaccharomyces pombe is a member of the RNase III family of double-strand-specific ribonucleases . To examine RNA structural features required for efficient cleavage by the Pac1 RNase, we tested a variety of double-stranded and hairpin RNAs as substrates for the enzyme . The Pac1 RNase required substrates that have a minimal helix length of about 20 base pairs . The enzyme cut both strands of the helix at sites separated by two base pairs . However, Pac1 was also able to make a single-stranded cleavage within an internal bulge of an authentic Escherichia coli substrate at the same site chosen by RNase III . Pac1 efficiently degraded the structurally complex adenovirus VA RNA(I), but was inactive against the short HIV-1 TAR RNA hairpin . These results indicate that the Pac1 RNase prefers straight, perfect helices, but it can tolerate internal bulges that do not distort the helix severely . Like its homologue from Saccharomyces cerevisiae, the Pac1 RNase cleaved at two in vivo RNA processing sites in a hairpin structure in the 3' external transcribed spacer of the S . pombe pre-rRNA, suggesting a role for the enzyme in rRNA maturation.

FEBS Lett, 1997 Sep 22, 415(1), 101 - 8
Isoform specificity of activators and inhibitors of protein kinase C gamma and delta; Keenan C et al.; Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol . Biol . Cell 5 (1994) 907-920) . We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening . This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma . We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

J Biol Chem, 1997 Oct 10, 272(41), 25851 - 5
RNA polymerase II subunits 2, 3, and 11 form a core subassembly with DNA binding activity; Kimura M et al.; RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide . We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni2+ affinity chromatography . After stepwise dissociation of the Rpb1- and Rpb3-tagged RNA polymerases fixed on Ni2+-resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained . Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the alpha2beta complex in the assembly of Escherichia coli RNA polymerase . Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.

J Mol Biol, 1997 Oct 3, 272(4), 509 - 22
A new Holliday junction resolving enzyme from Schizosaccharomyces pombe that is homologous to CCE1 from Saccharomyces cerevisiae; Whitby MC et al.; The resolution of Holliday junctions is a critical stage in recombination . We describe the identification and initial biochemical characterisation of a new Holliday junction resolvase from Schizosaccharomyces pombe . Resolvase activity was initially detected in partially purified cell-free extracts of S . pombe . Resolution of X-junction DNA occurred by the introduction of symmetrical cuts in strands of the same polarity . All cuts occurred 3' of thymine nucleotides with a possible preference for cleavage one nucleotide 3' from the point of strand crossover . During the course of these studies, a potential S . pombe homologue of the Saccharomyces cerevisiae Cruciform Cutting Endonuclease I was identified in the database (SpCCE1) . The gene was cloned by PCR, overexpressed in Escherichia coli and its product purified as a His-tagged fusion protein . Purified SpCCE1 binds to X-junctions in a structure-specific manner and resolves them to nicked linear duplex products that are repairable by DNA ligase . SpCCE1 cuts X-junctions in precisely the same way as the resolvase activity from partially purified extracts of S . pombe, indicating that they are probably the same . Finally, we show that SpCCE1 can function as a Holliday junction resolvase in vivo by its ability to complement a resolvase-deficient strain of E . coli .

Mol Cell Biol, 1997 Nov, 17(11), 6246 - 54
The fission yeast protein p73res2 is an essential component of the mitotic MBF complex and a master regulator of meiosis; Ayte J et al.; Depending on environmental conditions, Schizosaccharomyces pombe can remain in the stationary phase or enter into either premitotic or premeiotic DNA synthesis . This decision point is known as Start . In the mitotic cell cycle, regulation of G1/S-specific gene expression is dependent upon the MBF (Mlu1 binding factor) complex, known to contain p85cdc10 and p72res1 . Here we demonstrate that p73res2 controls cell cycle progression via its participation in the MBF complex, interacting directly with both p85cdc10 and p72res1 . In contrast, when cells enter into meiosis, the MBF complex is disrupted, and p73res2 shifts its regulatory function towards the transactivation of genes required for meiotic progression . These observations suggest that p73res2 plays a pivotal role at Start and constitutes an example of a transcription factor involved in the control of both mitotic and meiotic progression.

Genomics, 1997 Oct 1, 45(1), 211 - 5
Genomic organization and functional analysis of the murine protein phosphatase 1c gamma (Ppp1cc) gene; Okano K et al.; Protein phosphatase 1 holoenzymes are composed of catalytic subunits in combination with various regulatory subunits . In rodents, four different catalytic isoforms are known, PP1c alpha, -delta, -gamma 1, and -gamma 2 . Here we describe the genomic organization of the murine Ppp1cc gene that encodes the PP1c gamma 1 and PP1c gamma 2 isoforms . We determined that Ppp1cc maps to F1.2-G1.2 on chromosome 5 by FISH mapping . Southern hybridization and analysis of cross-hybridizing genomic clones revealed four Ppp1cc-related pseudogenes in the mouse genome . The authentic Ppp1cc gene encodes two isoforms, PP1c gamma 1 and PP1c gamma 2, that arise from alternative splicing and differ by retention of the last intron . The introns of Ppp1cc are flanked by short direct repeats, the significance of which is not clear . Both isoforms retain phosphatase function since they are able to complement the cold-sensitive PP1 defect caused by the dis2-11 mutation in the fission yeast Schizosaccharomyces pombe.

Curr Genet, 1997 Sep, 32(3), 244 - 6
Identification of the ure1+ gene encoding urease in fission yeast; Tange Y et al.; Cloning and sequencing of the ure1+ gene of Schizosaccharomyces pombe indicated that it encodes the urease which had been biochemically identified . The fission yeast urease has a one-subunit structure like those from plants but different from bacterial ureases which are composed of two or three distinct subunits . Genetic analyses showed that the ure1+ gene product is actually involved in urea metabolism.

Cancer Surv, 1997, 29, 25 - 45
S phase damage sensing checkpoints in mammalian cells; Larner JM et al.; Mammalian cells have evolved multiple responses for dealing with DNA damage . One response is to acutely downregulate DNA synthesis at the initiation step . Essentially nothing is known about the initial signal that activates this SDS pathway or the macromolecules involved in transducing the signal into the final inhibitory step at origins . Determining whether any radiation induced changes in known proteins involved in cell cycle regulation or in other signal transduction pathways are primary or secondary responses to DNA damage constitutes a major challenge to identifying members of the pathway . It may turn out to be easier to identify the final mediator in the pathway, namely the protein(s) whose interaction with origins is ultimately affected by radiation . Hopefully, mutations in SDS genes in genetically tractable systems such as S cerevisiae or Schizosaccharomyces pombe will allow the identification of homologous genes in mammals . Most tumour cells are TP53 negative, and yet it is not clear that TP53 status influences radiation sensitivity . The SDS pathway may therefore represent an important protective mechanism that stands in the way of effective tumour cell killing by radiation therapy . It is hoped that an understanding of this pathway will provide opportunities for developing novel antineoplastic targets and/or radiation sensitizers.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4331 - 7
Identification and characterization of a telomerase activity from Schizosaccharomyces pombe; Lue NF et al.; A telomerase-like primer extension activity has been detected in chromatographic fractions derived from Schizosaccharomyces pombe extracts . This primer extension activity acts preferentially on dG-rich oligodeoxynucleotides, is sensitive to RNase A pretreatment and requires all four deoxynucleotides for optimal polymerization . The extension products are also truncated by the inclusion of any one of the four dideoxynucleotides, consistent with the presence of all four bases in the S.pombe telomeric repeats . The intensity distribution of the extension products and the dideoxynucleotide termination pattern suggest that nucleotide addition is template directed, and that telomere-like sequences are added to the primers . In particular, the sequence d(CGGTTA), a variant of the S.pombe telomeric repeat, can be added directly by the in vitro activity . Partially purified S.pombe telomerase sediments as a 35S particle, suggesting that it exists in vivo as part of a large multi-protein complex.

J Bacteriol, 1997 Oct, 179(20), 6325 - 34
Rapamycin specifically interferes with the developmental response of fission yeast to starvation; Weisman R et al.; Rapamycin is a microbial macrolide which belongs to a family of immunosuppressive drugs that suppress the immune system by blocking stages of signal transduction in T lymphocytes . In Saccharomyces cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 stage of the cell cycle . Rapamycin is also an effective antifungal agent, affecting the growth of yeast and filamentous fungi . Unexpectedly, we observed that rapamycin has no apparent effect on the vegetative growth of Schizosaccharomyces pombe . Instead, the drug becomes effective only when cells experience starvation . Under such conditions, homothallic wild-type cells will normally mate and undergo sporulation . In the presence of rapamycin, this sexual development process is strongly inhibited and cells adopt an alternative physiological option and enter stationary phase . Rapamycin strongly inhibits sexual development of haploid cells prior to the stage of sexual conjugation . In contrast, the drug has only a slight inhibitory effect on the sporulation of diploid cells . A genetic approach was applied to identify the signal transduction pathway that is inhibited by rapamycin . The results indicate that either rapamycin did not suppress the derepression of sexual development of strains in which adenylate cyclase was deleted or the cyclic AMP-dependent protein kinase encoded by pka1 was mutated . Nor did rapamycin inhibit the unscheduled meiosis observed in pat1-114 mutants . Overexpression of ras1+, an essential gene for sexual development, did not rescue the sterility of rapamycin-treated cells . However, expression of the activated allele, ras1Val17, antagonized the effect of rapamycin and restored the ability of the cells to respond to mating signals in the presence of the drug . We discuss possible mechanisms for the inhibitory effect of rapamycin on sexual development in S . pombe.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11244 - 9
DNA polymerase delta isolated from Schizosaccharomyces pombe contains five subunits; Zuo S et al.; DNA polymerase delta (pol delta) plays an essential role in DNA replication, repair, and recombination . We have purified pol delta from Schizosaccharomyces pombe more than 10(3)-fold and demonstrated that the polymerase activity of purified S . pombe pol delta is completely dependent on proliferating cell nuclear antigen and replication factor C . SDS/PAGE analysis of the purified fraction indicated that the pol delta complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa . Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively . The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides . The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S . pombe chromosome 1.

Gene, 1997 Sep 1, 196(1-2), 165 - 74
Gene organization and protein sequence of the small subunits of Schizosaccharomyces pombe RNA polymerase II; Sakurai H et al.; RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe contains 10 different species of polypeptides . Previously, we cloned and sequenced both cDNA and the genes encoding the four large subunits, Rpb1, Rpb2, Rpb3 and Rpb5 . Later, other groups isolated the genes for Rpb6 and Rpb12 and cDNA for Rpb10 . Here, we cloned both cDNA and the genes encoding four small subunits, Rpb7, Rpb8, Rpb10 and Rpb11 . These genes were found to encode Rpb7, Rpb8, Rpb10 and Rpb11 consisting of 172 (19,103 Da), 125 (14,300 Da), 71 (8276 Da) and 123 (14,127 Da) amino acid residues, respectively . All these four subunits are homologous to the corresponding subunits of Saccharomyces cerevisiae RNA polymerase II . The rpb7 gene contains one intron, whereas the rpb8, rpb10 and rpb11 genes contain two introns . Taken altogether, the gene organization and the predicted protein sequence have been determined for all 10 subunits of the S . pombe RNA polymerase II.

EMBO J, 1997 Oct 15, 16(20), 6162 - 70
Multiple modes of activation of the stress-responsive MAP kinase pathway in fission yeast; Samejima I et al.; The Schizosaccharomyces pombe wis1(+) gene is essential for cell survival under stress conditions . The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition . We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo . Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal . Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues . Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.

Mol Cell Biol, 1997 Oct, 17(10), 5876 - 87
Multiple regulatory domains on the Byr2 protein kinase; Tu H et al.; Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe . Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1 . Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain . Ste4 and Ras1 interact with the regulatory domain of Byr2 directly . Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system . Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling . In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation . Furthermore, we provide evidence that Shk1, the S . pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

EMBO J, 1997 Oct 1, 16(19), 5977 - 87
Fission yeast Cut2 required for anaphase has two destruction boxes; Funabiki H et al.; The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation . Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction . This proteolysis depends on the APC (Anaphase-Promoting Complex)-cyclosome which contains ubiquitin ligase activity . The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box . We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis . Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations . Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not . Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes . Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.

EMBO J, 1997 Sep 15, 16(18), 5722 - 9
Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe; Remacle JE et al.; Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain . The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box . The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts . Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S . pombe when tested in a proximal TATA box context . Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position . Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position . Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

Amino Acid, Molecular Microbiology, Antibiotic, Microbes, Bioreactor, Microbe, Fermentation, Bacterium, Bioengineering, Microorganisms, Genome, Bacteriology, Activated Sludge, Microorganism, t, Growth Medium, f, Microbiology, g, Environmental Microbiology, d, Bacteria, p, Genetics, Bioassay, Gram positive, e, Fermentation, t, Prokaryotes, y, Microflora, b, Bacterial, b, E. coli, e, Ciprofloxacin, b, Neisseria, s, E. coli, p, Streptococcal, b, Escherichia coli, s, Enterobacters, m, Lactobacillus, b, Antibiotics, b, Cell suspensions, s, Bacterium, h, Corynebacterium, a, Salmonella typhimurium, c, Bacillus, s, Pseudomonas, a, Yeasts, b, Haemophilus, m, Salmonella, b, Yeasts, x, Enterobacteriacea, c, Bacillus subtilis, b, Yeasts, c, S. aureus, s, Lactobacillus, Pichia,

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005